Allelic diversity of phytoene synthase gene influences the transcription level in citrus fruit among a citrus F1 hybrid population.

Phytoene synthase (PSY) is one of the key regulatory enzyme on the biosynthesis and accumulation of carotenoid in citrus fruits. The transcriptional diversity of PSY is mainly attributed to the structural variation in promoter region among PSY alleles. In aim to clarify how this transcriptional diversity is regulated among them, PSY alleles responsible for carotenoid biosynthesis in the fruits are characterized and their promoter sequences were compared. Based on gene structure and expression pattern of PSY homologues on the clementine mandarin genome sequence, PSY alleles responsible for carotenoid biosynthesis are derived from a single locus in the scaffold 6. AG mapping population possessed four PSY alleles derived from parent lines of A255 and G434, and their F1 individuals with PSY-g2 allele tended to have low transcription level. From sequence comparison of their promoter regions, the cis-motif alternation from MYBPZM to RAV1AAT might be a candidate to influence the transcription level. Among the ancestral pedigree varieties of AG mapping population, the transcription level of PSY correlated with genotypes of MYBPZM and RAV1AAT motifs in the promoter region of PSY alleles, so that homozygous genotype of MYBPZM showed higher transcription level while heterozygous genotype of MYBPZM and RAV1AAT showed lower transcription level.

High serum carotenoids are associated with lower risk for developing elevated serum alanine aminotransferase among Japanese subjects: the Mikkabi cohort study.

Many recent studies have shown that antioxidant vitamins and/or carotenoids may reduce liver disease, but this association has not been well established with thorough longitudinal cohort studies. The objective of this study was to longitudinally investigate whether serum carotenoids at baseline are associated with the risk of developing elevated serum alanine aminotransferase (ALT) among Japanese subjects. We conducted a follow-up study of 1073 males and females aged between 30 and 79 years at baseline from the Mikkabi prospective cohort study. Those who participated in the baseline study and completed follow-up surveys were examined longitudinally. Exclusions included excessive alcohol consumption (≥60 g alcohol/d), hepatitis B and C and having a history of medication use for liver disease. A cohort of 213 males and 574 females free of elevated serum ALT (>30 IU/ml) at baseline was studied. Over a mean follow-up period of 7·4 (sd 3·1) years, thirty-one males and forty-nine females developed new elevated serum ALT. After adjustments for confounders, the hazard ratios for elevated serum ALT in the highest tertiles of basal serum ß-carotene, ß-cryptoxanthin and total provitamin A carotenoids against the lowest tertiles were 0·43 (95 % CI 0·22, 0·81), 0·51 (CI 0·27, 0·94) and 0·52 (CI 0·28, 0·97), respectively. For α-carotene and lycopene, borderline reduced risks were also observed; however, these were not significant. Our results further support the hypothesis that antioxidant carotenoids, especially provitamin A carotenoids, might help prevent earlier pathogenesis of non-alcoholic liver disease in Japanese subjects.

Diversity in the carotenoid profiles and the expression of genes related to carotenoid accumulation among citrus genotypes.

Carotenoids are not only important to the plants themselves but also are beneficial to human health. Since citrus fruit is a good source of carotenoids for the human diet, it is important to study carotenoid profiles and the accumulation mechanism in citrus fruit. Thus, in the present paper, we describe the diversity in the carotenoid profiles of fruit among citrus genotypes. In regard to carotenoids, such as ß-cryptoxanthin, violaxanthin, lycopene, and ß-citraurin, the relationship between the carotenoid profile and the expression of carotenoid-biosynthetic genes is discussed. Finally, recent results of quantitative trait locus (QTL) analyses of carotenoid contents and expression levels of carotenoid-biosynthetic genes in citrus fruit are shown.

High serum carotenoids associated with lower risk for the metabolic syndrome and its components among Japanese subjects: Mikkabi cohort study.

Recent epidemiological studies show the association of carotenoids with the metabolic syndrome (MetS), but thorough longitudinal cohort studies regarding this association have not been well conducted. The objective of this study was to investigate longitudinally whether serum carotenoids are associated with the risk of developing the MetS and its components in Japanese subjects. We conducted a follow-up study on 1073 men and women aged 30-79 years at the baseline from the Mikkabi prospective cohort study. Those who participated in the baseline and completed follow-up surveys were examined longitudinally. Over the 10-year period, 910 subjects (295 men and 615 women) took part in the follow-up survey at least once. Over a mean follow-up period of 7·8 (sd 2·9) years, thirty-six men and thirty-one women developed new MetS. After adjustments for confounders, the hazard ratio (HR) for the MetS in the highest tertile of serum ß-carotene against the lowest tertile was 0·47 (95 % CI 0·23, 0·95). On the other hand, significantly lower risks for dyslipidaemia were observed in the highest tertiles of serum α- and ß-carotene and ß-cryptoxanthin (HR 0·66; 95 % CI 0·46, 0·96; HR, 0·54; 95 % CI 0·37, 0·79; and HR 0·66; 95 % CI 0·44, 0·99, respectively). Other significant associations between the risks for obesity, high blood pressure and hyperglycaemia with serum carotenoids were not observed. Our results further support the hypothesis that eating a diet rich in carotenoids might help prevent the development of the MetS and its complications in Japanese subjects.

Expression and functional analysis of two lycopene ß-cyclases from citrus fruits.

In the present study, two LCYb genes (CitLCYb1 and CitLCYb2) were isolated from Satsuma mandarin (Citrus unshiu Marc.), Valencia orange (Citrus sinensis Osbeck) and Lisbon lemon (Citrus limon Burm.f.) and their functions were analyzed by the color complementation assay in lycopene-accumulating E. coli cells. The results showed that CitLCYb1 and CitLCYb2 shared high identity at the amino acid level among the three citrus varieties. The N-terminal region of the two proteins encoded by CitLCYb1 and CitLCYb2 was predicted to contain a 51-residue chloroplastic transit peptide, which shared low similarity. In Satsuma mandarin, the secondary structures of the CitLCYb1 and CitLCYb2 encoding proteins without the transit peptide were quite similar. Moreover, functional analysis showed that both enzymes of CitLCYb1 and CitLCYb2 participated in the formation of ß-carotene, and when they were co-expressed with CitLCYe, α-carotene could be produced from lycopene in E. coli cells. However, although CitLCYb2 could convert lycopene to α-carotene in E. coli cells, its extremely low level of expression indicated that CitLCYb2 did not participate in the formation of α-carotene during the green stage in the flavedo. In addition, the high expression levels of CitLCYb1 and CitLCYb2 during the orange stage played an important role in the accumulation of ß,ß-xanthophylls in citrus fruits. The results presented in this study might contribute to elucidate the mechanism of carotenoid accumulation in citrus fruits.

Regulation of carotenoid accumulation and the expression of carotenoid metabolic genes in citrus juice sacs in vitro.

In the present study, to investigate the mechanisms regulating carotenoid accumulation in citrus, a culture system was set up in vitro with juice sacs of three citrus varieties, Satsuma mandarin (Citrus unshiu Marc.), Valencia orange (Citrus sinensis Osbeck), and Lisbon lemon (Citrus limon Burm.f.). The juice sacs of all the three varieties enlarged gradually with carotenoid accumulation. The changing patterns of carotenoid content and the expression of carotenoid metabolic genes in juice sacs in vitro were similar to those ripening on trees in the three varieties. Using this system, the changes in the carotenoid content and the expression of carotenoid metabolic genes in response to environmental stimuli were investigated. The results showed that carotenoid accumulation was induced by blue light treatment, but was not affected by red light treatment in the three varieties. Different regulation of CitPSY expression, which was up-regulated by blue light while unaffected by red light, led to different changes in carotenoid content in response to these two treatments in Satsuma mandarin and Valencia orange. In all three varieties, increases in carotenoid content were observed with sucrose and mannitol treatments. However, the accumulation of carotenoid in the two treatments was regulated by distinct mechanisms at the transcriptional level. With abscisic acid (ABA) treatment, the expression of the genes investigated in this study was up-regulated in Satsuma mandarin and Lisbon lemon, indicating that ABA induced its own biosynthesis at the transcriptional level. This feedback regulation of ABA led to decreases in carotenoid content. With gibberellin (GA) treatment, carotenoid content was significantly decreased in the three varieties. Changes in the expression of genes related to carotenoid metabolism varied among the three varieties in response to GA treatment. These results provided insights into improving carotenoid content and composition in citrus during fruit maturation.

Synergistic interaction of cigarette smoking and alcohol drinking with serum carotenoid concentrations: findings from a middle-aged Japanese population.

Previous studies have indicated low serum carotenoid concentrations among cigarette smokers and/or alcohol drinkers, but little is known about the interaction of smoking and drinking with serum carotenoids. We tested the hypothesis that smoking and drinking reduce serum carotenoid concentrations synergistically. A total of 1073 subjects (357 male and 716 female) who had received health examinations in the town of Mikkabi, Shizuoka Prefecture, Japan, participated in the study. The subjects were divided into six groups according to alcohol intake (non-drinkers, < 1 g/d; light drinkers, > or = 1, < 25 g/d; moderate-to-heavy drinkers, > or = 25 g/d) and smoking status (non-smokers and current smokers). The dietary intakes and serum concentrations of six carotenoids (lycopene, alpha-carotene, beta-carotene, lutein, beta-cryptoxanthin and zeaxanthin) within each group were evaluated cross-sectionally. The dietary intakes of all carotenoids did not differ in the six groups after adjusting for age and sex. The multivariate-adjusted means of the serum carotenoid concentrations in non-drinkers did not differ between non-smokers and current smokers. In contrast, the adjusted means of serum alpha-carotene, beta-carotene and beta-cryptoxanthin were significantly lower than those with increased alcohol intake, and these lower serum carotenoids among alcohol drinkers were more evident in current smokers than in non-smokers. Serum lycopene of moderate-to-heavy drinkers was significantly lower than that of non-drinkers, but it was not influenced by smoking. Neither smoking nor drinking was associated with the serum concentrations of lutein and zeaxanthin. These results suggest that smoking and drinking may reduce the serum alpha-carotene, beta-carotene and beta-cryptoxanthin concentrations in a synergistic manner.

Associations of serum carotenoid concentrations with the metabolic syndrome: interaction with smoking.

Recent epidemiological studies show the associations of serum antioxidant status with the metabolic syndrome. Oxidative stress may play an important role in the pathogenesis of diabetes and CVD. Actually, smoking is a potent oxidative stressor in man, but little is known about the interaction of serum carotenoids and the metabolic syndrome with smoking status. In this study, the associations of the serum carotenoids with the metabolic syndrome stratified by smoking habit were evaluated cross-sectionally. A total of 1073 subjects (357 male and 716 female) who had received health examinations in the town of Mikkabi, Shizuoka Prefecture, Japan, participated in the study. Among total subjects, the OR for the metabolic syndrome in the highest tertile of serum beta-carotene was 0.41 (95 % CI 0.18, 0.92) after adjusting confounders. In current smokers, significantly lower OR were observed in the middle (OR 0.10; 95 % CI 0.01, 0.72) and highest (OR 0.06; 95 % CI 0.01, 0.73) tertiles of serum beta-carotene. Furthermore, lower OR were observed in accordance with tertiles of serum alpha-carotene and beta-cryptoxanthin in current smokers (P for trend 0.042 and 0.036, respectively). In contrast, in non-smokers, a significantly lower OR was observed in the highest tertile of serum beta-carotene (OR 0.30; 95 % CI 0.10, 0.89) after multiple adjustment. Inverse associations of serum carotenoids with the metabolic syndrome were more evident among current smokers than non-smokers. These results support that antioxidant carotenoids may have a protective effect against development of the metabolic syndrome, especially in current smokers who are exposed to a potent oxidative stress.

Abscisic acid affects expression of citrus FT homologs upon floral induction by low temperature in Satsuma mandarin (Citrus unshiu Marc.).

After a long juvenile period, citrus trees undergo seasonal flowering cycles. Under natural conditions, citrus flowering is regulated mainly by low ambient temperatures around 15-20 °C and water deficit stress. Recent studies have revealed that fluctuations in the expression of citrus homologs of FLOWERING LOCUS T (FT, encoding a flowering integrator) are correlated with their presumed role as flower-promoting signals. Previous ectopic expression analyses have demonstrated the flower-promoting function of citrus FT homologs. In this study, we examined whether abscisic acid (ABA) affects the expression of FT homologs and the flowering induced by low ambient temperatures. Application of exogenous ABA to potted Satsuma mandarin (Citrus unshiu Marc.) trees resulted in transient accumulation of citrus FT homolog transcripts. The promoter of one citrus FT homolog, CiFT3, was active in transgenic A. thaliana (Arabidopsis thaliana) and responded to exogenous and endogenous ABA. CiFT3 is preferentially expressed in shoots, and its expression was affected by flower-inductive treatments. Endogenous ABA accumulated in mandarin shoots during the floral induction period at 15 °C and under field conditions. The accumulation of ABA was correlated with the accumulation of FT homolog transcripts and flowering intensity. It was consistent with changes in the expression of genes related to ABA metabolism. The abundance of carotenoid precursors that serve as substrates for ABA biosynthesis decreased in leaves during the accumulation of ABA. Our data indicate that ABA and carotenoid precursors in leaves influence the flowering of mandarin trees induced by low temperature.

Increased CiFT abundance in the stem correlates with floral induction by low temperature in Satsuma mandarin (Citrus unshiu Marc.).

After several years in the juvenile phase, adult citrus trees show seasonal periodicity of flowering. A prolonged exposure to low temperature is one of the most important environmental cues for floral induction in citrus. In the present study, the expression of flowering-related genes during the annual cycle of flowering and inductive low-temperature treatment in Satsuma mandarin (Citrus unshiu Marc.) trees is investigated. Simultaneously, floral induction, which occurs before the period of morphological flower development, was estimated as the number of flowers after the forcing of sprouting by defoliation at 25 degrees C. The expression of citrus FLOWERING LOCUS T homologues, CiFT, showed a seasonal increase during the floral induction period and was also induced by an artificial low-temperature treatment (15 degrees C) at which floral induction occurred. By contrast, the mRNA level of CiFT did not show any distinct changes following a warm-temperature treatment (25 degrees C) for 2.5 months, during which time floral induction was completely suppressed. Changes in the expression of the citrus homologues of TERMINAL FLOWER 1, LEAFY, and APETALA1 did not show any correlation with floral induction in the field or under artificial low-temperature conditions. In juvenile seedlings of Satsuma mandarin, which does not flower even under inductive low-temperature conditions, the mRNA levels of CiFT were not affected by the low-temperature treatment, unlike adult tissues. These results suggest that low temperature promotes floral induction via the activation of CiFT transcription in adult Satsuma mandarin trees and that, in the juvenile plant, CiFT transcription does not respond to low temperature.

Quantification of carotenoids in citrus fruit by LC-MS and comparison of patterns of seasonal changes for carotenoids among citrus varieties.

To quantify the 18 carotenoids on the basic routes of the carotenoid biosynthesis in plants simultaneously, a method for liquid chromatography-mass spectrometry (LC-MS) using atmospheric pressure chemical ionization was developed. With this method, the seasonal changes of carotenoids in the flavedo and juice sacs of 39 citrus varieties were analyzed. On the basis of the patterns of seasonal changes of carotenoids in both flavedo and juice sacs, 39 citrus varieties were classified. In flavedo, 39 varieties were classified into 5 clusters, in which the carotenoid profiles were carotenoid-poor, phytoene-abundant, violaxanthin-abundant, violaxanthin- and beta-cryptoxanthin-abundant, and phytoene-, violaxanthin-, and beta-cryptoxanthin-abundant, respectively. In juice sacs, they were classified into 4 clusters, in which the carotenoid profiles were carotenoid-poor, violaxanthin-abundant, violaxanthin- and phytoene-abundant, and violaxanthin-, phytoene-, and beta-cryptoxanthin-abundant, respectively. In flavedo, many citrus varieties, except for the carotenoid-poor and phytoene-abundant varieties, massively accumulated beta,epsilon-carotenoids (e.g., lutein), beta,beta-carotenoids (e.g., beta-cryptoxanthin and violaxanthin), and phytoene, in that order. In juice sacs, the accumulation order among beta,beta-carotenoids was observed. Violaxanthin accumulation preceded beta-cryptoxanthin accumulation in violaxanthin-, phytoene-, and beta-cryptoxanthin-abundant varieties. In each variety, the carotenoid profiles of the flavedo and juice sacs on the basis of the concentration in violaxanthin and beta-cryptoxanthin were similar, with the exception of a few varieties.

Carotenoid accumulation in Japanese apricot (Prunus mume Siebold & Zucc.): molecular analysis of carotenogenic gene expression and ethylene regulation.

To elucidate the regulatory mechanisms of carotenogenesis in Japanese apricot (Prunus mume Siebold & Zucc.), the relationships between carotenoid accumulation and the expression of the carotenogenic genes, phytoene synthase (PmPSY-1), phytoene desaturase (PmPDS), zeta-carotene desaturase (PmZDS), lycopene beta-cyclase (PmLCYb), lycopene epsilon-cyclase (PmLCYe), beta-carotene hydroxylase (PmHYb), and zeaxanthin epoxidase (PmZEP), were analyzed in two cultivars with different ripening traits, 'Orihime' and 'Nanko.' In 'Orihime' fruits, large amounts of carotenoids accumulated on the tree, concomitant with the induction of PmPSY-1 and the downstream carotenogenic genes PmLCYb, PmHYb, and PmZEP. In 'Nanko' fruits, carotenoids accumulated mainly after harvest, correlating with an appreciable induction of PmPSY-1 expression, but the downstream genes were not notably induced, which may explain the lower total carotenoid content in 'Nanko' than in 'Orihime.' In both cultivars, a decrease in PmLCYe expression and increased or constant PmLCYb expression could cause the metabolic shift from beta,epsilon-carotenoid synthesis to beta,beta-carotenoid synthesis that occurs as ripening approaches. Next, the effects of ethylene on the expression of PmPSY-1 and carotenoid accumulation were investigated in 'Nanko' fruits treated with propylene or 1-methylcyclopropene (1-MCP). Propylene treatment induced both ethylene production and carotenoid accumulation. PmPSY-1 was constitutively expressed, but propylene treatment accelerated its induction. 1-MCP treatment caused a slight inhibition of carotenoid accumulation along with the repression, although not complete, of PmPSY-1. Collectively, although PmPSY-1 expression was not exclusively regulated by ethylene, both the notable induction of PmPSY-1 accelerated by ethylene and the subsequent induction of the downstream carotenogenic genes, especially PmLCYb, could be necessary for the massive carotenoid accumulation that occurs during ripening. Furthermore, the switch from PmLCYe expression to PmLCYb expression could cause beta,beta-carotenoid accumulation in both Japanese apricot cultivars.

Serum carotenoid concentrations are inversely associated with serum aminotransferases in hyperglycemic subjects.

Hyperglycemia increases the generation of free radicals by glucose auto-oxidation, and the increment of free radicals may lead to liver cell damage. In this study, we tested the hypothesis that hyperglycemia-induced increases of serum liver enzymes among its physiological concentration would be inversely associated with serum antioxidant carotenoid level. Study subjects were 857 male and female Japanese who had received health examinations in 2003. Those with a history of liver disease and excessive alcohol drinkers were excluded. The associations of serum six-carotenoid concentrations with serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) stratified by glucose tolerance status were evaluated cross-sectionally. Serum AST and ALT concentrations in the groups of impaired fasting glucose (IFG) and diabetes were significantly higher than those in the normal group. The multivariate-adjusted means of the serum AST and ALT concentrations in IFG and diabetes group were significantly low in accordance with the tertiles of the serum beta-cryptoxanthin and beta-carotene concentrations. The most inverse association of serum liver enzyme and carotenoid concentration was observed in beta-cryptoxanthin. Antioxidant carotenoid, especially beta-cryptoxanthin, may act a deterrent substance against increasing the serum aminotransferase in the earlier pathogenesis of liver dysfunction among hyperglycemic subjects.

High Vitamin C Intake with High Serum ß-Cryptoxanthin Associated with Lower Risk for Osteoporosis in Post-Menopausal Japanese Female Subjects: Mikkabi Cohort Study.

Recent epidemiological studies show that antioxidant vitamins and carotenoids might be beneficial to the maintenance of bone health. Recently, we found that serum carotenoids were inversely associated with the risk of developing osteoporosis in post-menopausal Japanese female subjects. However, little is known about the vitamin alone and/or the combination of the vitamin and carotenoid with the risk of osteoporosis. The objective of this study was to investigate longitudinally whether antioxidant vitamins and their combination with carotenoids are associated with the risk of developing of osteoporosis. We conducted a follow-up study on 187 post-menopausal female subjects from the Mikkabi prospective cohort study. Those who participated in previous bone mineral density (BMD) surveys and completed four years of follow-up were examined longitudinally. During a four-year follow-up, fifteen of the post-menopausal female subjects developed new-onset osteoporosis. After adjustment for confounders, the odds ratios (OR) for osteoporosis in the highest tertiles of vitamins C and E and retinol intakes against the lowest tertiles were 0.15 (95% confidence interval (CI): 0.02-0.99), 0.50 (CI: 0.08-3.23), and 1.49 (CI: 0.36-6.22), respectively. Furthermore, a significantly lower odds ratio was observed in the higher vitamin C intake group (169-625 mg/d) with higher serum ß-cryptoxanthin (1.88-10.53 µM) against the lower vitamin C intake group (47-168 mg/d) with lower serum ß-cryptoxanthin (0.24-1.84 µM) used for the reference group (p<0.05). The combination of ß-cryptoxanthin and vitamin C is inversely associated with the risk of developing osteoporosis in post-menopausal Japanese female subjects.

Overexpression of a citrus basic helix-loop-helix transcription factor (CubHLH1), which is homologous to Arabidopsis activation-tagged bri1 suppressor 1 interacting factor genes, modulates carotenoid metabolism in transgenic tomato.

To explore the transcription factors associated with carotenoid metabolism in citrus fruit, one transcription factor (CubHLH1) was selected through microarray screening in Satsuma mandarin (Citrus unshiu Marc.) fruit, which was treated with exogenous ethylene or gibberellin (GA), accelerating or retarding carotenoid accumulation in peel, respectively. The amino acid sequence of CubHLH1 has homology to Arabidopsis activation-tagged bri1 suppressor 1 (ATBS1) interacting factor (AIF), which is functionally characterized as a negative regulator of the brassinolide (BR) signalling pathway. Yeast two-hybrid analysis revealed that protein for CubHLH1 could interact with Arabidopsis and tomato ATBS1. Overexpression of CubHLH1 caused a dwarf phenotype in transgenic tomato (Solanum lycopersicum L.), suggesting that CubHLH1 has a similar function to Arabidopsis AIF. In the transgenic tomato fruit at ripening stage, the lycopene content was reduced along with the changes in carotenoid biosynthetic gene expression. The abscisic acid (ABA) content of all the transgenic tomato fruit was higher than that of the wild type. These results implied that CubHLH1 is considered to have a similar function to Arabidopsis AIFs and might be directly involved in carotenoid metabolism in mature citrus fruit.

High-serum carotenoids associated with lower risk for developing type 2 diabetes among Japanese subjects: Mikkabi cohort study.

OBJECTIVE: Recent epidemiological studies show the association of antioxidant carotenoids with type 2 diabetes, but thorough longitudinal cohort studies regarding this association have not been well conducted. The objective of this study was to investigate longitudinally whether serum carotenoids are associated with the risk for developing type 2 diabetes among Japanese subjects. RESEARCH DESIGN AND METHODS: We conducted a follow-up study on 1073 males and females aged 30-79 years at the baseline from the Mikkabi prospective cohort study. Those who participated in the baseline and completed follow-up surveys were examined longitudinally. Over the 10-year period, 910 subjects (295 males and 615 females) took part in the follow-up survey at least one time. A cohort of 264 males and 600 females free of diabetes at baseline was studied. RESULTS: Over a mean follow-up period of 7.8 years (SD=2.9), 22 males and 33 females developed new type 2 diabetes. After adjustments for confounders, the HRs for type 2 diabetes in the highest tertiles of serum α-carotene, ß-cryptoxanthin, and total provitamin A carotenoids against the lowest tertiles were 0.35 (95% CI 0.15 to 0.82), 0.43 (CI 0.20 to 0.92) and 0.41 (CI 0.19 to 0.90), respectively. For ß-carotene and zeaxanthin, borderline reduced risks were also observed, but these were not significant. CONCLUSIONS: Our results further support the hypothesis that eating a diet rich in carotenoids, especially provitamin A carotenoids, might help prevent the development of type 2 diabetes in Japanese patients. TRIAL REGISTRATION NUMBER: NIFT-2013001.

Effect of blue LED light intensity on carotenoid accumulation in citrus juice sacs.

In the present study, the effects of blue LED light intensity on carotenoid accumulation and expression of genes related to carotenoid biosynthesis were investigated in the juice sacs of Satsuma mandarin (Citrus unshiu Marc.) and Valencia orange (Citrus sinensis Osbeck) in vitro. The results showed that 100 µmol m(-2)s(-1) blue LED light (100B) was effective for increasing carotenoid content, especially ß-cryptoxanthin, in Satsuma mandarin after cultured in vitro for four weeks. In Valencia orange, in contrast, 50 µmol m(-2)s(-1) blue LED light (50B) treatment was effective for inducing carotenoid accumulation through increasing the contents of two major carotenoids, all-trans-violaxanthin and 9-cis-violaxanthin. In addition, gene expression results showed that the simultaneous increases in the expression of genes (CitPSY, CitPDS, CitZDS, CitLCYb2, and CitHYb) involved in producing ß,ß-xanthophylls were well consistent with the accumulation of ß-cryptoxanthin in Satsuma mandarin under 100B, and violaxanthin in Valencia orange under 50B. The results presented herein contribute to further elucidating the regulatory mechanism of carotenoid accumulation by blue LED light.

Regulation of ascorbic acid metabolism by blue LED light irradiation in citrus juice sacs.

In the present study, the effects of red and blue LED lights on the accumulation of ascorbic acid (AsA) were investigated in the juice sacs of three citrus varieties, Satsuma mandarin, Valencia orange, and Lisbon lemon. The results showed that the blue LED light treatment effectively increased the AsA content in the juice sacs of the three citrus varieties, whereas the red LED light treatment did not. By increasing the blue LED light intensity, the juice sacs of the three citrus varieties accumulated more AsA. Moreover, continuous irradiation with blue LED light was more effective than pulsed irradiation for increasing the AsA content in the juice sacs of the three citrus varieties. Gene expression results showed that the modulation of AsA accumulation by blue LED light was highly regulated at the transcription level. The up-regulation of AsA biosynthetic genes (CitVTC1, CitVTC2, CitVTC4, and CitGLDH), AsA regeneration genes (CitMDAR1, CitMDAR2, and CitDHAR) and two GSH-producing genes (CitGR and CitchGR) contributed to these increases in the AsA content in the three citrus varieties.

Identification and quantification of the conjugated metabolites derived from orally administered hesperidin in rat plasma.

Hesperidin is a biologically effective flavonoid. Several studies have reported that dietary hesperidin was converted to conjugated metabolites, such as hesperetin-glucuronides and sulfoglucuronides, during absorption and metabolism. However, the chemical structures of the conjugated metabolites, especially the sites of glucuronidation and sulfoglucuronidation in plasma, were unconfirmed. Therefore, the concentrations of the metabolites conjugated at various sites in plasma could not be individually quantified. In the present study, we identified the chemical structures and concentrations of the major conjugated metabolites in rat plasma after oral administration of hesperidin. Two hesperetin-glucuronides were prepared and identified as hesperetin-7-O-beta-D-glucuronide and hesperetin-3'-O-beta-D-glucuronide. Using these authentic compounds, the concentrations of hesperetin-7-O-beta-D-glucuronide and hesperetin-3'-O-beta-D-glucuronide in rat plasma were individually determined by liquid chromatography-mass spectrometry. In rat plasma, hesperetin-glucuronides were primarily comprised of hesperetin-7-O-beta-D-glucuronide and hesperetin-3'-O-beta-D-glucuronide. The concentration of hesperetin-7-O-beta-D-glucuronide was slightly higher than that of hesperetin-3'-O-beta-D-glucuronide. Furthermore, not only hesperetin conjugates but also homoeriodictyol conjugates were observed in rat plasma. The present study is the first report elucidating the chemical structures and changes in individual concentrations in rat plasma of glucuronides derived from orally administered hesperidin.

Multiple linear regression analysis of the seasonal changes in the serum concentration of beta-cryptoxanthin.

Beta-cryptoxanthin (beta-CRX) is a carotenoid pigment found in Satsuma mandarin (Citrus unshiu Marc.) fruit, which is heavily produced in Japan. In this study, we evaluated the seasonal changes in the serum beta-CRX level and investigated predictors of serum beta-CRX level by multiple linear regression analysis. Blood tests and self-administered questionnaires were used every other month for one year. The subjects were healthy volunteers, 15 males and 12 females. The serum beta-CRX levels increased dramatically as the intake of Satsuma mandarin increased; the maximum increase was noted in January. Multiple linear regression analysis showed that, in males, the serum beta-CRX level could be predicted by Satsuma mandarin intake, age and the month of blood sampling; however, it was inversely associated with alcohol and smoking habits. Conversely, in females, the serum beta-CRX concentration could be predicted by Satsuma mandarin intake, the month of blood sampling and age; however, it was inversely associated with body mass index. The results of multiple linear regression analysis suggest that the serum beta-CRX levels can be used to evaluate the intake volume of Satsuma mandarin. Furthermore, beta-CRX is a useful biomarker to estimate the beneficial effects of Satsuma mandarin intake in epidemiological studies.