Effects of phosphorus on fruit soluble sugar and citric acid accumulations in citrus.

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.

The effect of ß-cyclocitral treatment on the carotenoid content of transgenic Marsh grapefruit (Citrus paradisi Macf.) suspension-cultured cells.

This work reports the development of suspension culture system of transgenic Marsh grapefruit (Citrus paradisi Macf., Rutaceae) callus overexpressing bacterial phytoene synthase; and the use of this suspension culture to investigate the effects of ß-cyclocitral on carotenoid content and composition. At a ß-cyclocitral concentration of 0.5 mM and after ten days cultivation, analysis of the carotenoids showed a significant increase in the content of ß-, α-carotene, and phytoene predominantly. The maximal increase in total provitamin A carotenoids content following ß-cyclocitral application was ~2-fold higher than the control, reaching 245.8 µg/g DW. The trend for increased transcript levels of biosynthetic genes PSY and ZDS correlated with the enhancement of the content of these carotenes following ß-cyclocitral treatment and GC-MS based metabolite profiling showed significant changes of metabolite levels across intermediary metabolism. These findings suggest that ß-cyclocitral can act as a chemical elicitor, to enhance the formation of carotenes in citrus suspension-cultured cells (SCC), which could be utilized in studying the regulation of carotenoid biosynthesis and biotechnological application to the renewable production of nutritional carotenoids.

Glycemic index, glycemic load, and glycemic response to pomelo in patients with type 2 diabetes.

Food intake has a great influence on blood glucose in patients with diabetes. This study was to determine the glycemic index (GI) and glycemic load (GL) of a particular pomelo named Majia pomelo and its effects on postprandial glucose (PPG) in patients with type 2 diabetes (T2D). Twenty healthy subjects and 20 T2D patients (controlled on lifestyle measures and/or metformin) were tested on 2 separate days with 50 g of glucose and 50 g equivalent of carbohydrates from Majia pomelo for GI measurement. To test effects of Majia pomelo on PPG, 19 hospitalized T2D patients (controlled on insulin therapy) were selected for a 9-day study. The dose of insulin for each patient was adjusted on the first 3 days. A total of 100 mg Majia pomelo was consumed per meal in the last 3 tested days. Blood glucose was measured to evaluate the glycemic excursions. The GIs for Majia pomelo in healthy individuals and T2D patients were 78.34±1.88 and 72.15±1.95 respectively. The value of GL was as low as 4.23 in diabetic patients with serving size of 100 g pomelo, indicting Majia pomelo as a high GI but low GL fruit. Consumption of Majia pomelo in hospitalized T2D patients did not cause significant glucose fluctuation. It was concluded that high GI pomelo can serve as a low GL fruit if it is consumed with a limited daily amount and thus can be supplied to diabetic patients. These results may mean more varieties of food choices for T2D patients.

Dynamic changes in methylome and transcriptome patterns in response to methyltransferase inhibitor 5-azacytidine treatment in citrus.

DNA methylation is known to play an important role in various developmental processes in plants. However, there is a general lack of understanding about the possible functions of DNA methylation in fruit trees. Using callus as a model, methylome, transcriptome and metabolite changes were assessed after treatment with the DNA methyltransferase inhibitor 5-azacytidine (5azaC). Genome-wide methylome analysis revealed the demethylation of a diverse of genes, including many genes encoding transcription factors (TFs), genes involved in biological processes, and the up-regulation of a wide range of transposable elements (TEs). Combined with the RNA-seq data, we observed no obvious genome-wide correlation between the changes in methylation status and expression levels. Furthermore, 5azaC treatment induced carotenoid degradation along with strong activation of carotenoid cleavage dioxygenases 1 (CpCCD1). Functional complementation analysis in bacterial system showed that CpCCD1 exhibited strong catalytic activities toward zeaxanthin, ß-carotene and lycopene. In summary, 5azaC treatments induced carotenoid degradation by CpCCD1 activation and led to a genome-wide demethylation effect.

Genome-wide identification and functional analysis of S-RNase involved in the self-incompatibility of citrus.

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.

Exogenous γ-aminobutyric acid treatment affects citrate and amino acid accumulation to improve fruit quality and storage performance of postharvest citrus fruit.

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.

Salicylic acid treatment reduces the rot of postharvest citrus fruit by inducing the accumulation of H2O2, primary metabolites and lipophilic polymethoxylated flavones.

To comprehensively analyze the effects of salicylic acid (SA) on the storability of Satsuma mandarin (Citrus unshiu), fruits were treated with 2mM SA. The disease incidence of control/SA-treated fruit at 50d and 120d after treatment was 23.3%/10% and 67.3%/23.3%, respectively, suggesting that SA treatment can significantly reduce the rot rate of postharvest citrus fruit. Fruit quality assays revealed that the treatment can maintain fruit firmness without affecting the inner quality. Furthermore, the contents of H2O2 and some defense-related metabolites, such as ornithine and threonine, in citrus pericarp, were significantly increased by SA treatment. Moreover, it was lipophilic polymethoxylated flavones, rather than flavanone glycosides, that accumulated in SA-treated fruits and these can directly inhibit pathogen development. These results suggest that the effects of SA on postharvest citrus fruit may be attributed to the accumulation of H2O2 and defense-related metabolites.

Sweating treatment enhances citrus fruit disease resistance by inducing the accumulation of amino acids and salicylic acid-induced resistance pathway.

To clarify the mechanism of fruit disease resistance activated by sweating treatment, 'Guoqing NO.1' Satsuma mandarin (Citrus unshiu Marc.) fruits were treated by sweating, which is a traditional prestorage treatment in China. Subsequently, we performed inoculation and physiological characterization, two-dimensional gel electrophoresis (2-DE) proteomics analysis and metabonomics analysis based on gas chromatography coupled to mass spectrometry (GC-MS) and high-performance liquid chromatography/electrospray ionization-time of flight-mass spectrometry (HPLC-qTOF-MS). The results showed that sweating treatment significantly inhibited pathogen infection without negatively affecting the fruit commercial quality. In addition, sweating treatment rapidly promoted the accumulation of amino acids (such as proline and serine). Meanwhile, hydrogen peroxide (H2 O2 ) and salicylic acid (SA) were significantly accumulated in the sweating-treated fruit. Thereafter, some stress-response proteins and metabolites [such as ascorbate peroxidase (APX), ß-1,3-glucanase, vanillic acid and rutin] which can be induced by SA were also significantly increased in the sweating-treated fruit. Taken together, the disease resistance induced by sweating treatment might be attributed to: (1) the induction of the accumulation of amino acids; and (2) the accumulation of SA and subsequent activation of SA-induced resistance pathway, which can induce the stress-response proteins and metabolites that can directly inhibit pathogen development.

A novel macromolecular extract screened from satsuma with pro-inflammatory effect.

Excessive consumption of horticultural fruit is a double-edged sword with both positive and negative effects. In Eastern countries, a large number of people have suffered from shang huo as a result of excessive consumption of "heating" foods, such as lychee, longan, mandarin orange, mango and civet durian. The present study adopted a step by step strategy screened the compositions with pro-inflammatory effect in satsuma fruits. The pro-inflammatory effects of all fractions were evaluated in RAW 264.7 cell lines by enzyme-linked immunosorbent assay (ELISA) and RT-PCR tests. The soluble water extract (SWE) from satsuma increased the production of prostaglandin E2 (PGE2) and promoted the expression level of cyclooxygenase-2 (COX-2) mRNA. SWE and high molecular weight molecules extracted from soluble water extract (HSWE) were respectively fractionated by dialysis bags and gel filtration chromatography. The macromolecular fraction named F1 was further obtained from HSWE, and could increase the production of inflammatory mediators. Finally F1 was resolved by SDS-PAGE and six proteins were identified by mass spectrometry. Compared with other detected proteins, polygalacturonase inhibitor (PGIP) and chitinase were the most likely candidate pro-inflammatory proteins according to molecular mass, and both of them were Citrus unshiu species. cDNA sequences of PGIP and chitinase were cloned and their functions were predicted as defensive proteins by SMART analysis. Excessive intake of these defensive proteins may result in adverse food reactions in human beings, such as shang huo and other immune responses.

Anti-inflammatory effect of auraptene extracted from trifoliate orange (Poncirus trifoliate) on LPS-stimulated RAW 264.7 cells.

Poncirus trifoliate is a traditional Chinese medicinal plant used for treating inflammation-related diseases for a long time and trifoliate orange contains abundant auraptene. The present study was to evaluate auraptene as a potential anti-inflammatory agent and investigate the mechanism of auraptene against prostaglandins E2 (PGE2) and cyclooxygenase-2 (COX-2) on lipopolysaccharide (LPS)-stimulated RAW 264.7 cells by comparing it with aspirin as a positive control group. The methods of enzyme-linked immunosorbent assay, reverse transcriptive polymerase chain reaction, real-time PCR, and western-blotting were used in the study. The results showed that auraptene exhibited better biocompatibility and lower cytotoxicity. At the same time, it significantly inhibited the production of PGE2 on LPS-stimulated macrophage cells. The auraptene-treated group had a higher COX-2 mRNA expression but relatively lower COX-2 protein level which implied that auraptene suppressed the post-transcriptional expression of COX-2 protein but not the transcriptional process. Compared with aspirin, the lower cytotoxicity of auraptene can make it a potential source for medicine that can benefit patients who are suffering from chronic inflammatory diseases and need long-term medication.

Comparative proteomic and metabolomic profiling of citrus fruit with enhancement of disease resistance by postharvest heat treatment.

BACKGROUND: From field harvest to the consumer's table, fresh citrus fruit spends a considerable amount of time in shipment and storage. During these processes, physiological disorders and pathological diseases are the main causes of fruit loss. Heat treatment (HT) has been widely used to maintain fruit quality during postharvest storage; however, limited molecular information related to this treatment is currently available at a systemic biological level. RESULTS: Mature 'Kamei' Satsuma mandarin (Citrus unshiu Marc.) fruits were selected for exploring the disease resistance mechanisms induced by HT during postharvest storage. Proteomic analyses based on two-dimensional gel electrophoresis (2-DE), and metabolomic research based on gas chromatography coupled to mass spectrometry (GC-MS), and liquid chromatography quadrupole time-of-flight mass spectrometry (LC-QToF-MS) were conducted. The results show resistance associated proteins were up-regulated in heat treated pericarp, such as beta-1, 3-glucanase, Class III chitinase, 17.7 kDa heat shock protein and low molecular weight heat-shock protein. Also, redox metabolism enzymes were down-regulated in heat treated pericarp, including isoflavone reductase, oxidoreductase and superoxide dismutase. Primary metabolic profiling revealed organic acids and amino acids were down-regulated in heat treated pericarp; but significant accumulation of metabolites, including tetradecanoic acid, oleic acid, ornithine, 2-keto-d-gluconic acid, succinic acid, turanose, sucrose, galactose, myo-inositol, glucose and fructose were detected. Noticeably, H2O2 content decreased, while, lignin content increased in heat treated pericarp compared to the control, which might increase fruit resistibility in response to external stress. Also, flavonoids, substances which are well-known to be effective in reducing external stress, were up-regulated in heat treated pericarp. CONCLUSIONS: This study provides a broad picture of differential accumulation of proteins and metabolites in postharvest citrus fruit, and gives new insights into HT improved fruit disease resistance during subsequent storage of 'Kamei' Satsuma mandarin. Interpretation of the data for the proteins and metabolites revealed reactive oxygen species (ROS) and lignin play important roles in heat treatment induced fruit resistance to pathogens and physiological disorders.

Volatile constituents of wild citrus Mangshanyegan (Citrus nobilis Lauriro) peel oil.

Volatiles of a wild mandarin, Mangshanyegan (Citrus nobilis Lauriro), were characterized by GC-MS, and their aroma active compounds were identified by aroma extract dilution analysis (AEDA) and gas chromatography-olfactometry (GC-O). The volatile profile of Mangshanyegan was compared with those of other four citrus species, Kaopan pummelo (Citrus grandis), Eureka lemon (Citrus limon), Huangyanbendizao tangerine (Citrus reticulata), and Seike navel orange (Citrus sinensis). Monoterpene hydrocarbons predominated in Mangshanyegan, in particular d-limonene and ß-myrcene, which accounted for 85.75 and 10.89% of total volatiles, respectively. Among the 12 compounds with flavor dilution factors (FD) = 27, 8 oxygenated compounds, including (Z)- and (E)-linalool oxides, were present only in Mangshanyegan. The combined results of GC-O, quantitative analysis, odor activity values (OAVs), and omission tests revealed that ß-myrcene and (Z)- and (E)-linalool oxides were the characteristic aroma compounds of Mangshanyegan, contributing to the balsamic and floral notes of its aroma.

The Or gene enhances carotenoid accumulation and stability during post-harvest storage of potato tubers.

Provitamin A carotenoids in staple crops are not very stable during storage and their loss compromises nutritional quality. To elucidate the fundamental mechanisms underlying carotenoid accumulation and stability, we investigated transgenic potato tubers that expressed the cauliflower Orange (Or) gene. We found that the Or transgene not only promoted retention of ß-carotene level, but also continuously stimulated its accumulation during 5 months of cold storage. In contrast, no increased levels of carotenoids were observed in the tubers of vector-only controls or a yellow-flesh variety during the same period of storage. The increased carotenoid accumulation was found to be associated with the formation of lipoprotein-carotenoid sequestering structures, as well as with the enhanced abundance of phytoene synthase, a key enzyme in the carotenoid biosynthetic pathway. Furthermore, the provitamin A carotenoids stored were shown to be stable during simulated digestion and accessible for uptake by human intestinal absorptive cells. Proteomic analysis identified three major functional groups of proteins (i.e. heat shock proteins, glutathione-S-transferases, and carbohydrate metabolic proteins) that are potentially important in the Or-regulated carotenoid accumulation. Our results show that regulation of carotenoid sequestration capacity is an important mechanism by which carotenoid stability is regulated. Our findings suggest that induction of a proper sink structure formation in staple crops may provide the crops with a unique ability to promote and/or stabilize provitamin A accumulation during plant growth and post-harvest storage.

Comparison of cell wall metabolism in the pulp of three cultivars of 'Nanfeng' tangerine differing in mastication trait.

BACKGROUND: Like sweet orange (Citrus sinensis), tangerine (Citrus reticulata) is another citrus crop grown widely throughout the world. However, whether it shares a common mechanism with sweet orange in forming a given mastication trait is still unclear. In this study, three 'Nanfeng' tangerine cultivars, 'Yangxiao-26' ('YX-26') with inferior mastication trait, elite 'YX-26' with moderate mastication trait and 'Miguang' ('MG') with superior mastication trait, were selected to investigate the formation mechanism of mastication trait. RESULTS: 'MG' had the lowest contents of total pectin, protopectin and lignin and the highest gene expression levels of citrus polygalacturonase (PG) and pectin methylesterase (PME) at the end of fruit ripening, whereas 'YX-26' had the lowest water-soluble pectin (WSP) content, the highest lignin content and the lowest PG and PME expression levels. The contents of cellulose and hemicellulose were similar among the three tangerines. CONCLUSION: The fruit mastication trait of C. reticulata was determined by the proportions of WSP and protopectin as well as lignin content, not by cellulose and hemicellulose contents. Pectin content could be a major contribution to the feeling of mastication trait, while PG and PME exhibited an important role in forming a given mastication trait according to the present results as well as previous results for C. sinensis.

Physicochemical and molecular analysis of cell wall metabolism between two navel oranges (Citrus sinensis) with different mastication traits.

BACKGROUND: FJ72-1 navel orange and its bud mutant FJWC exhibit differences in melting texture character which are influenced mostly by cell wall metabolism. Here we compared the contents of water soluble pectin (WSP), protopectin, total pectin (TP), cellulose, and hemicellulose, activities of polygalaturonase (PG), pectin methylesterase (PME), pectate lyases (PL), cellulase (Cel) and gene expression levels of PG, PME, PL and Cel between the two cultivars. RESULTS: The content of cellulose and hemicellulose decreased progressively during fruit ripening. At the harvest time (230 DAF), the content of cellulose and hemicellulose in FJWC were obviously higher than those in FJ72-1; the WSP content, PG activities and its gene expression level in FJWC was lower than those in FJ72-1. Moreover, gene expression levels of PME and Cel in FJWC were only one-quarter of those in FJ72-1 at 230 DAF. CONCLUSION: The present work showed that the inferior melting character of FJWC attributed to the lower WSP, higher TP or protopectins, higher cellulose and hemicellulose than those in the pulp of FJ72-1 at harvest time. Lower expression levels of PG, PME and Cel at harvest time might be associated with the inferior melting character.

Discovery and comparative profiling of microRNAs in a sweet orange red-flesh mutant and its wild type.

BACKGROUND: Red-flesh fruit is absent from common sweet orange varieties, but is more preferred by consumers due to its visual attraction and nutritional properties. Our previous researches on a spontaneous red-flesh mutant revealed that the trait is caused by lycopene accumulation and is regulated by both transcriptional and post-transcriptional mechanisms. However, the knowledge on post-transcriptional regulation of lycopene accumulation in fruits is rather limited so far. RESULTS: We used Illumina sequencing method to identify and quantitatively profile small RNAs on the red-flesh sweet orange mutant and its wild type. We identified 85 known miRNAs belonging to 48 families from sweet orange. Comparative profiling revealed that 51 known miRNAs exhibited significant expression differences between mutant (MT) and wild type (WT). We also identified 12 novel miRNAs by the presence of mature miRNAs and corresponding miRNA*s in the sRNA libraries. Comparative analysis showed that 9 novel miRNAs are differentially expressed between WT and MT. Target predictions of the 60 differential miRNAs resulted 418 target genes in sweet orange. GO and KEGG annotation revealed that high ranked miRNA-target genes are those implicated in transcription regulation, protein modification and photosynthesis. The expression profiles of target genes involved in carotenogenesis and photosynthesis were further confirmed to be complementary to the profiles of corresponding miRNAs in WT and MT. CONCLUSION: This study comparatively characterized the miRNAomes between the red-flesh mutant and the wild type, the results lay a foundation for unraveling the miRNA-mediated molecular processes that regulate lycopene accumulation in the sweet orange red-flesh mutant.

Functional characterization of Citrus PSY gene in Hongkong kumquat (Fortunella hindsii Swingle).

Citrus, rich in carotenoids, is the most important fruit crop based on the total annual production. In the carotenoid biosynthesis pathway, phytoene synthase (PSY, EC 2.5.1.32) catalyzes the dimerization of two molecules of geranylgeranyl pyrophosphate (GGPP) to phytoene and has been shown to be a rate-limiting enzyme for the synthesis of carotenoids. In this study, we investigated catalytic activity of CsPSY from Cara Cara navel orange (Citrus sinensis Osbeck) by heterologous expression in Escherichia coli containing a GGPP-producing plasmid. Moreover, the effects of CsPSY overexpression on carotenoid accumulation were also functionally analyzed in transgenic Hongkong kumquat (Fortunella hindsii Swingle). The resulting transgenic plants produced orange fruits, and extracts from the fruits of four overexpressing plants had a 2.5-fold average increase of phytoene with the content approximately 71.38 microg/g fresh weight. Lycopene, beta-carotene, and beta-cryptoxanthin in transgenic fruits were also markedly increased, whereas the levels of lutein and violaxanthin kept nearly unchanged with 1.1-1.3 folds variation. Transcript levels of carotenoid biosynthetic genes in the CsPSY overexpressed plants remained unaltered except that PDS and ZDS showed a minor increase. This study suggests that CsPSY plays a crucial role in citrus carotenoid biosynthesis and could be used as a means of engineering fruit crop for the production of carotenoids.

RAPD-based genetic analysis of offsprings from the sexual cross using allotetraploid citrus somatic hybrid as pollen parent.

Thirty-one polymorphic decamer primers were selected to genotype 92 progenies from the cross between Yiben No.4, a monoembryonic diploid F1 hybrid of Citrus reticulata Blanco cv Huanongbendizao tangerine and C. ichangensis Swingle, and [Hamlin sweet orange + Rough lemon], an allotetraploid somatic hybrid of Citrus sinensis Osbeck cv. Hamlin and C. jambhiri Lush cv. Rough Lemon. chi2 (Chi-square) analysis of RAPD markers in the progenies indicated they were randomly transmitted from the four donor parents, without significant difference between the diploids and triploids. However, these progenies were clustered into three major groups using dendrogram constructed by UPGMA, skewed to three parents in certain degrees, 15 (13 triploids and 2 diploids) to Hamlin, 16 (9 and 7) to Yiben No. 4, and 61 (57 and 4) to [Hamlin sweet orange + Rough Lemon] from which genomic contribution was predominant in progenies, respectively.

Lycopene-epsilon-cyclase pre-mRNA is alternatively spliced in Cara Cara navel orange (Citrus sinensis Osbeck).

Lycopene-epsilon-cyclase is one of the key enzymes related to alpha-carotene metabolism in plants. A full-length cDNA of 1300 bp encoding lycopene-epsilon-cyclase (Lyce) was generated from Cara Cara navel orange, a unique navel orange containing both lycopene and beta-carotene in its pulp, with little or no alpha-carotene. The gene had a 14 bp nucleotides deletion and caused a terminal mutation. DNA sequence corresponding to the deletion region revealed that two repeats of 6 bp (AGGTGT) were flanking the region in both Cara Cara and its original variety, Washington navel oranges, but a 2 bp (AT) insertion was only found in Cara Cara which explain the alternative splicing character of the gene.