Hikaru Matsumoto

Accumulation of Carotenoids and Expression of Carotenoid Biosynthetic Genes during Maturation in Citrus Fruit

The relationship between carotenoid accumulation and the expression of carotenoid biosynthetic genes during fruit maturation was investigated in three citrus varieties, Satsuma mandarin (Citrus unshiu Marc.), Valencia orange (Citrus sinensis Osbeck), and Lisbon lemon (Citrus limon Burm.f.). We cloned the cDNAs for phytoene synthase (CitPSY), phytoene desaturase (CitPDS), ζ-carotene (car) desaturase (CitZDS), carotenoid isomerase (CitCRTISO), lycopene β-cyclase (CitLCYb), β-ring hydroxylase (CitHYb), zeaxanthin (zea) epoxidase (CitZEP), and lycopene ϵ-cyclase (CitLCYe) from Satsuma mandarin, which shared high identities in nucleotide sequences with Valencia orange, Lisbon lemon, and other plant species. With the transition of peel color from green to orange, the change from β,ϵ-carotenoid (α-car and lutein) accumulation to β,β-carotenoid (β-car, β-cryptoxanthin, zea, and violaxanthin) accumulation was observed in the flavedos of Satsuma mandarin and Valencia orange, accompanying the disappearance of CitLCYe transcripts and the increase in CitLCYb transcripts. Even in green fruit, high levels of β,ϵ-carotenoids and CitLCYe transcripts were not observed in the juice sacs. As fruit maturation progressed in Satsuma mandarin and Valencia orange, a simultaneous increase in the expression of genes (CitPSY, CitPDS, CitZDS, CitLCYb, CitHYb, and CitZEP) led to massive β,β-xanthophyll (β-cryptoxanthin, zea, and violaxanthin) accumulation in both the flavedo and juice sacs. The gene expression of CitCRTISO was kept low or decreased in the flavedo during massive β,β-xanthophyll accumulation. In the flavedo of Lisbon lemon and Satsuma mandarin, massive accumulation of phytoene was observed with a decrease in the transcript level for CitPDS. Thus, the carotenoid accumulation during citrus fruit maturation was highly regulated by the coordination of the expression among carotenoid biosynthetic genes. In this paper, the mechanism leading to diversity in β,β-xanthophyll compositions between Satsuma mandarin and Valencia orange was also discussed on the basis of the substrate specificity of β-ring hydroxylase and the balance of expression between upstream synthesis genes (CitPSY, CitPDS, CitZDS, and CitLCYb) and downstream synthesis genes (CitHYb and CitZEP).

The Homeostasis Model Assessment-insulin Resistance Index Is Inversely Associated with Serum Carotenoids in Non-diabetic Subjects

BACKGROUND Carotenoids may reduce the risk for diabetes mellitus, but little is known about the association of insulin resistance with serum carotenoids in non-diabetic subjects. This study aimed to investigate whether the homeostasis model assessment-insulin resistance (HOMA-IR) index would be lower in the presence of high serum carotenoid concentrations in non-diabetic subjects. METHODS A total of 812 subjects (256 males and 556 females) who had received health examinations in 2003 participated in the study. The associations of the serum-carotenoid concentrations and HOMA-IR were evaluated cross-sectionally. The multivariate-adjusted geometric means of HOMA-IR by the tertiles of the serum carotenoid concentration were calculated after adjusting for age, body mass index, systolic blood pressure, total cholesterol, triacylglycerols, current tobacco use, regular alcohol intake, exercise habits and total energy intake. Associations among high HOMA-IR (3.0+ mU×mmol/L2) across tertiles of serum carotenoid concentration were assessed by tests for logistic regression analysis. RESULTS In male subjects, the multivariate adjusted geometric mean of HOMA-IR was inversely associated with the serum β-cryptoxanthin concentrations. In female subjects, an inverse association of the serum carotenoid concentration and HOMA-IR was observed in lycopene, β-cryptoxanthin, and zeaxanthin. The confounding factor-adjusted odds ratios (OR) for high HOMA-IR on the highest tertiles of serum α-carotene, β-carotene, β-cryptoxanthin, and zeaxanthin were 0.18 [95% confidence interval (CI): 0.06-0.52], 0.22 (95% CI: 0.07-0.67), 0.34 (95% CI: 0.12-0.96), and 0.30 (95% CI: 0.11-0.79), respectively, in male subjects. On the other hand, in female subjects, the adjusted OR for high HOMA-IR on the highest tertiles of serum lycopene and β-cryptoxanthin were 0.39 (95% CI: 0.21-0.73) and 0.51 (95% CI: 0.28-0.95), respectively. CONCLUSIONS The serum antioxidant carotenoids were inversely associated with HOMA-estimated insulin resistance in non-diabetic subjects.

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.

Effect of Postharvest Temperature and Ethylene on Carotenoid Accumulation in the Flavedo and Juice Sacs of Satsuma Mandarin (Citrus unshiu Marc.) Fruit

The effect of postharvest temperature (5, 20, and 30 degrees C) and ethylene at different temperatures (20 and 5 degrees C) on carotenoid content and composition and on the expression of the carotenoid biosynthesis-related genes was investigated in the flavedo and juice sacs of Satsuma mandarin ( Citrus unshiu Marc.) fruit. Under an ethylene-free atmosphere, storage at 20 degrees C rapidly increased the carotenoid content in flavedo and maintained the content in juice sacs. In contrast, storage at 5 and 30 degrees C gradually decreased the content in juice sacs but slowly increased that in flavedo. Under an ethylene atmosphere, storage at 20 degrees C enhanced the carotenoid accumulation in flavedo more dramatically than found under an ethylene-free atmosphere with distinct changes in the carotenoid composition but did not noticeably change the content and composition in juice sacs. In contrast, storage at 5 degrees C under an ethylene atmosphere repressed carotenoid accumulation with changes in the carotenoid composition in flavedo but did not clearly change the carotenoid content in juice sacs. Under an ethylene-free atmosphere, differences in the gene expression profile among the temperatures were observed but were not well-correlated with those in the carotenoid content in flavedo and juice sacs. Under an ethylene atmosphere, in flavedo, the gene expression of phytoene synthase (PSY) and phytoene desaturase (PDS) was slightly higher at 20 degrees C but lower at 5 degrees C than under an ethylene-free atmosphere. At 20 degrees C, the gene expression of several carotenoid biosynthetic enzymes promoted by ethylene seemed to be responsible for the enhanced accumulation of carotenoid in flavedo. In contrast, at 5 degrees C, the repressed gene expression of PSY and PDS by ethylene seemed to be primarily responsible for the repressed accumulation of carotenoid in flavedo. In juice sacs, the small response of the gene expression to ethylene seemed to be responsible for small changes in carotenoid accumulation under an ethylene atmosphere.

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.

Effect of Blue and Red LED Light Irradiation on β-Cryptoxanthin Accumulation in the Flavedo of Citrus Fruits

β-Cryptoxanthin (β-cry), an antioxidant abundant in citrus fruits, plays an important role in the prevention and treatment of certain diseases, especially cancers. In the present study, to increase the content of β-cry in citrus flavedo, the effects of blue (470 nm) and red (660 nm) light-emitting diode (LED) lights on the accumulation of carotenoids and expression of genes related to carotenoid biosynthesis were investigated in the flavedo of Satsuma mandarin. The results showed that accumulation of β-cry was induced by red light, while it was not affected by blue light. The accumulation of β-cry under red light was attributed to simultaneous increases in the expression of CitPSY, CitPDS, CitZDS, CitLCYb1, CitLCYb2, CitHYb, and CitZEP. The results presented herein might provide new strategies to enhance the commercial and nutritional value of citrus fruits.

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.

Expression and functional analysis of two lycopene β-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 β-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 β,β-xanthophylls in citrus fruits. The results presented in this study might contribute to elucidate the mechanism of carotenoid accumulation in citrus fruits.

Effect of Different Postharvest Temperatures on the Accumulation of Sugars, Organic Acids, and Amino Acids in the Juice Sacs of Satsuma Mandarin (Citrus unshiu Marc.) Fruit

To elucidate the effect of different postharvest temperatures on the accumulation of sugars, organic acids, and amino acids and to determine the best temperature to minimize their postharvest change, their content after harvest was investigated at 5, 10, 20, and 30 °C for 14 days in the juice sacs of Satsuma mandarin (Citrus unshiu Marc. cv. Aoshima-unshiu) fruit. In all sugars, the changes were negligible at all temperatures. Organic acids decreased slightly at all temperatures, with the exception of malic acid at 30 °C, which increased slightly. Two amino acids, ornithine and glutamine, increased at 5 °C, but they did not increase at other temperatures. In 11 amino acids (phenylalanine, tryptophan, tyrosine, isoleucine, leucine, valine, threonine, lysine, methionine, histidine, and γ-amino butyric acid), the content was higher at 20 and 30 °C than at other temperatures. Thus, the content of amino acids was more variable than that of sugars and organic acids in response to temperatures. Moreover, amino acids responded to temperature differently: two amino acids were cold responsive, and 11 were heat-responsive. The best temperature to minimize the postharvest changes in amino acid profiles in the juice sacs of Aoshima-unshiu was 10 °C. The responsiveness to temperatures in two cold-responsive (ornithine and glutamine) and five heat-responsive (phenylalanine, tryptophan, valine, lysine, and histidine) amino acids was conserved among three different Satsuma mandarin cultivars, Aoshima-unshiu (late-maturing cultivar), Silverhill (midmaturing cultivar), and Miyagawa-wase (early-maturing cultivar). The metabolic responsiveness to temperature stress was discussed on the basis of the changes in the amino acid profile.

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 β-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 β,β-xanthophylls were well consistent with the accumulation of β-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.