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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.

The muscat flavor of table grapes often decreases after harvest, but the contributions of post-harvest temperature to the flavor loss were unknown. In the present study, the effect of different postharvest temperatures on muscat flavor and the content of aroma volatiles was investigated at 0, 2, 5, and 10 °C for 12 weeks in the berries of ‘Shine Muscat’ ( Vitis labruscana Baily × Vitis vinifera Vitis vinifera L.), which has a strong muscat flavor. Furthermore, after 2, 4, and 8 weeks of storage at 0 °C, the grapes were subjected to post-storage conditioning at 10 °C for 14 more days, and changes in the aroma volatile content were investigated. A sensory test showed that after 4 weeks of storage, the muscat flavor obviously decreased at 0 °C, but at 10 °C, the muscat flavor was maintained. Gas chromatography analysis of aroma volatiles showed that the content of linalool, which is one of the causative agents of muscat flavor, dramatically decreased at low temperatures (0, 2, and 5 °C) in comparison with that at 10 °C. During storage, the linalool content was much lower at 0 °C than at 10 °C in both the skin and the flesh. Interestingly, the linalool content, as well as muscat flavor, which decreased during storage at 0 °C, increased after the grapes were subjected to post-storage conditioning at 10 °C. The linalool content of the grape conditioned at 10 °C for 1–14 more days was higher than that of the grape continuously stored at 0 °C. These results indicated that muscat flavor is strongly influenced by postharvest temperature and that low-temperature storage enhances the loss of muscat flavor and the decrease in linalool content, but storage at 10 °C delayed and minimized them. Moreover, these results showed that post-storage conditioning at an increased temperature before consumption would be effective to increase the muscat flavor even after flavor loss during low-temperature storage at 0 °C.

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.

A brown skin spot developed on the berries of Japanese grape cultivar Shine Muscat during storage at 0 °C for 3 months. The fungus isolated from these skin spots was identified as Cadophora luteo-olivacea based on the sequence of its ribosomal DNA internal transcribed spacer region. Morphological and culture characteristics corresponded to those reported previously from C. luteo-olivacea; this fungus also reproduced the same symptoms after inoculation and was reisolated from the symptomatic spots. This is the first report of a postharvest disease of grape caused by C. luteo-olivacea.

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.

We developed 708 gene-based markers for citrus genome analysis. Sequence-tagged site (STS) primers were designed that were located in conserved exon regions and whose PCR products spanned genomic introns. Of these, 79.7 % comprised cleaved amplified polymorphic sequence markers. The gene-based markers and their annotation and position on Clementine scaffolds ver. 1.0 permitted comparison of the genetic map and the Clementine genome sequence. The 708 gene-based markers were used to construct a genetic map using the 87 progenies (AG population) from the cross between ‘Okitsu 46 gou’ (‘Sweet Spring’ (‘Ueda unshiu’ (Citrus unshiu) × Hassaku (Citrus hassaku Hort. ex Tanaka)) × ‘Trovita’ orange) × ‘Kankitsu Chukanbohon Nou 5 gou’ (‘Lee’ (Citrus clementina × tangelo) × Citrus kinokuni). The markers were integrated using common STSs on different phase maps in cross-pollination mode. The integrated map (AGI map) comprised 706 loci, including two morphological traits, and spanned 990.9 centimorgans (cM) with an average marker distance of 1.40 cM. These markers formed nine linkage groups (LGs) (corresponding to citrus physical chromosomes): LG-01 to LG-09 corresponded to Scaffold_01, Scaffold_07, Scaffold_09, Scaffold_06, Scaffold_03, Scaffold_02, Scaffold_04, Scaffold_08, and Scaffold_05, respectively. LG-08 and LG-09 contained morphological traits controlling embryo color and seedlessness. Eighty-eight loci comprised three or more alleles on the AGI map; 36.4 % of them were related to transcription factors and DNA-binding proteins. The 708 gene-based markers and the AGI map are valuable for integrating various citrus genetic maps, alignment of genomic sequences, chromosome assignment, and understanding the diversity of citrus germplasms.

Citrus fruits contain significant amounts of various carotenoids and some of them are known to benefit human health. Approximately 115 different carotenoids have been reported in citrus fruits, and the color of the fruit and peel are caused by carotenoid accumulation (Stewart and Wheaton, 1973). The carotenoid content and composition in

To learn how to regulate carotenoid content in citrus fruit, we studied the mechanism of carotenoid accumulation using physiological and genetic methods. Based on differences in the carotenoid profiles of flavedo and juice sacs, citrus species could be roughly divided into three groups: carotenoidpoor, violaxanthin-abundant, and b-cryptoxanthin-abundant groups. A comparison of the gene expression among several citrus species with different carotenoid profiles showed that the difference in carotenoid profiles among citrus species was highly regulated by coordination of the expression for genes related to carotenoid biosynthesis and catabolism. Quantitative trait loci related to carotenoid content were also identified to select progeny with high carotenoid content. Moreover, we showed that the effect of postharvest ethylene on carotenoid accumulation in flavedo varied with temperature. Under an ethylene atmosphere, in flavedo, carotenoid accumulation was enhanced more dramatically than under an ethylene-free atmosphere at 20°C but repressed at 5°C.

The effects of electrostatic atomization on ascorbate (AsA) metabolism in broccoli (Brassica oleracea L. var. italica) were studied and the possible molecular mechanisms discussed. Electrostatic atomization, delayed the yellowing process, and ethylene production and respiration rates were significantly suppressed in broccoli after harvest. The AsA content declined rapidly to a lower level in the controls after harvest, and the reduction of AsA was suppressed by the treatment with electrostatic atomization during the storage period. In addition, modulation of the AsA reduction by electrostatic atomization was highly regulated at the transcription level. Up-regulation of the AsA biosynthetic genes (BO-VTC1, BO-VTC2, and BO-GLDH), and AsA regeneration genes (BO-MDAR1, BO-MDAR2, and BO-DHAR) led to the suppression of AsA reduction in broccoli treated with electrostatic atomization after harvest. These results indicated that electrostatic atomization treatment might be a new effective approach for delaying the senescence of broccoli.

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.

We developed a 384 multiplexed SNP array, named CitSGA-1, for the genotyping of Citrus cultivars, and evaluated the performance and reliability of the genotyping. SNPs were surveyed by direct sequence comparison of the sequence tagged site (STS) fragment amplified from genomic DNA of cultivars representing the genetic diversity of citrus breeding in Japan. Among 1497 SNPs candidates, 384 SNPs for a high-throughput genotyping array were selected based on physical parameters of Illumina’s bead array criteria. The assay using CitSGA-1 was applied to a hybrid population of 88 progeny and 103 citrus accessions for breeding in Japan, which resulted in 73,726 SNP calls. A total of 351 SNPs (91 %) could call different genotypes among the DNA samples, resulting in a success rate for the assay comparable to previously reported rates for other plant species. To confirm the reliability of SNP genotype calls, parentage analysis was applied, and it indicated that the number of reliable SNPs and corresponding STSs were 276 and 213, respectively. The multiplexed SNP genotyping array reported here will be useful for the efficient construction of linkage map, for the detection of markers for marker-assisted breeding, and for the identification of cultivars.

To aid the breeding of citrus (Citrus sp.) for high carotenoid content, we assayed the fruit flesh of 48 cultivars and selections within a parental population consisting of both old and new cultivars and selections at two locations in Japan. The mean total carotenoid (CAR) content across all 48 cultivars and selections over the two locations was 26.59 μg·g−1 fresh weight (FW). The most prominent carotenoid was β-cryptoxanthin [BCR (12.09 μg·g−1 FW)] followed by violaxanthin [VIO (8.04 μg·g−1 FW)], ζ-carotene (2.27 μg·g−1 FW), phytoene (1.86 μg·g−1 FW), and β-carotene (0.96 μg·g−1 FW). Broad-sense heritabilities of CAR, BCR, and VIO were 0.80 or greater based on a sample of five fruit on one tree per location in one time sampling for 1 year in a location, which were revealed to be large enough for gauging the genetic variation. The mean CAR and BCR contents in a cultivar and selection group in advanced generations were nearly the same as in the initial population, suggesting no or little selection pressure on carotenoid content in the citrus breeding so far. High carotenoid contents in cultivars and selections released or selected recently, which have high fruit qualities, suggest their high potential for combining high fruit quality and high carotenoid content in breeding. We showed that the critical phenotypic value used in selecting hybrid seedlings can be determined from the estimate of environmental variance.

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.

The effects of 1-methylcyclopropene (1-MCP) on ascorbate (AsA) metabolism were studied and the possible molecular mechanisms were discussed for two cultivars of cauliflower (Brassica oleracea, L. var. botrytis), ‘Violet Queen’ and ‘Snow Crown’. Ethylene production was suppressed in ‘Violet Queen’, while it was increased in ‘Snow Crown’ by 1-MCP treatment. Meanwhile, the changing patterns of AsA metabolism after harvest were different between the two cauliflower cultivars. In ‘Violet Queen’, AsA content decreased in the control after harvest, and the loss of AsA was delayed by 1-MCP treatment. In ‘Snow Crown’, AsA content remained almost constant and was not affected by 1-MCP treatment. In ‘Violet Queen’, the gene expression of BO-APX1, BO-APX2, and BO-sAPX was down-regulated, while the gene expression of BO-DHAR and BO-GLDH was up-regulated by 1-MCP treatment. The regulation of these genes contributed to the suppression of AsA reduction in ‘Violet Queen’ treated by 1-MCP. In ‘Snow Crown’, simultaneous down-regulation of BO-APX1, BO-APX2, and BO-sAPX, which were responsible for AsA breakdown, and BO-MDAR1, BO-MDAR2, BO-DHAR, and BO-GLDH, which were responsible for AsA regeneration and biosynthesis, might lead to maintain the AsA level constant with 1-MCP treatment.

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.

The effects of 1-methylcyclopropene (1-MCP) on ascorbate (AsA) metabolism in two cultivars of broccoli ( Brassica oleracea L . var. italica ), ‘Haitsu’ and ‘Ryokurei’, were studied and the possible molecular mechanisms are discussed. The results showed that 1-MCP treatment delayed the yellowing and suppressed ethylene production. Meanwhile, the AsA content declined to a lower level in the control during storage, and the reduction of AsA was significantly suppressed by the treatment with 1-MCP in the two cultivars. Gene expression analyses by real-time PCR showed that 1-MCP treatment down-regulated the expression of BO-APX1 and BO-APX2 , and up-regulated that of BO-DHAR and BO-GLDH compared with the control. The regulation of this gene expression might contribute to the suppression of AsA reduction by the 1-MCP treatment in ‘Haitsu’ and ‘Ryokurei’ broccoli. The results arising from this study might provide new insights into the possible mechanism, by which treatment with 1-MCP delayed senescence.

Gibberellin 3 (GA 3 )-responsive genes were investigated with a citrus 22K oligoarray 1 to further the understanding of transcriptional regulation by GA 3 treatment in Satsuma mandarin fruit ( Citrus unshiu Marc.). 213 GA 3 -responsive genes were identified that showed a 3-fold or greater expression change after 72 h GA 3 treatment, compared to expression after 72 h air treatment. GA 3 treatment induced expression of pathogenesis-related (PR) proteins and genes that function in photosynthesis, chloroplast biogenesis, resistance, defense and stress. Also, GA 3 treatment reduced the transcription of several ethylene-inducible genes, such as carotenoid metabolic genes, which are associated with fruit ripening. Contrasting effects between GA 3 and ethylene were observed on photosynthesis and chloroplast biogenesis, chlorophyll metabolism, and carotenoid metabolism, indicating that the endogenous GA 3 level might be important for the endogenous regulation of maturation and senescence in mature citrus fruit. It was also found that the GA response pathway was likely to take part in cross-talk with the pathogen-related pathway in mature citrus fruit.

A comprehensive transcriptome analysis using a citrus 22K oligoarray was performed to identify ethylene-responsive genes and gain an understanding of the transcriptional regulation by ethylene in mandarin fruit (Citrus unshiu Marc.). In the 72 h after ethylene treatment, 1493 genes were identified as ethylene-responsive with more than 3-fold expression change, and an interesting aspect of gene regulation by ethylene was observed, namely, that more than half of the ethylene-responsive genes were repressed. This aspect might suggest that ethylene demotes numerous biological processes and plays an important role in fruit ripening and senescence. Ethylene repressed the transcription of most genes involved in photosynthesis, chloroplast biogenesis, and sugar metabolism, while it induced the transcription of several genes related to resistance, defense, stress, amino acid synthesis, protein degradation, and secondary metabolism. In carotenoid metabolism, the sensitivity and responsive patterns to exogenous ethylene were significantly different among carotenoid biosynthesis genes. Ethylene might cause a change of their transcriptional balance and influence carotenoid composition of fruits. Besides, most of ethylene biosynthesis genes and its signal transduction components did not show any significant expression change (

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 β-cryptoxanthin, violaxanthin, lycopene, and β-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.

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.

Citrus fruits are a rich source of carotenoids. cDNAs for carotenoid biosynthetic enzymes have been identified from their flavedos and juice sacs, and were used to examine expression patterns of carotenogenic genes during fruit development by several groups. However, functions of most of the gene products have not been verified yet. In this report, we examined catalytic activities of two carotenogenic enzymes from navel orange (Citrus sinensis Osbeck), phytoene desaturase (CitPds) and lycopene-β-cyclase (CitLcyb), and one enzyme from Satsuma mandarin (Citrus unshiu Marcow), lycopene-?-cyclase (CitLcye). We also conducted in vitro import assay of the three proteins along with two other carotenogenic enzyme from navel orange, phytoene synthase (CitPsy) and carotenoid β-ring hydroxylase (CitChyb), using isolated chloroplasts, and confirmed their plastid localization and the presence of transit peptides that were cleaved upon import. Furthermore, we examined their suborganellar localization. CitPsy was found to be peripherally associated with the membrane, while CitPds was mainly recovered in the soluble fraction. By contrast, CitLcyb and CitLcye were targeted both to the soluble and to the membrane compartments, although the latter showed a stronger association to the membrane than the former. Finally, CitChyb was exclusively inserted into the chloroplast internal membranes. These data should help us better understand the mechanism of carotenoid biosynthesis.

To investigate the relationship between a carotenoid profile and gene expression for carotenoid cleavage dioxygenases, three citrus varieties that exhibit different 9-cis-violaxanthin levels in their juice sacs, Satsuma mandarin (Citrus unshiu Marc.; a variety accumulating a low level of 9-cis-violaxanthin), Valencia orange (Citrus sinensis Osbeck; variety accumulating a high level of 9-cis-violaxanthin), and Lisbon lemon (Citrus limon Burm.f.; a variety accumulating an undetectable level of 9-cis-violaxanthin) were used. Three cDNAs (CitCCD1, CitNCED2, and CitNCED3) were cloned. The recombinant CitCCD1 protein cleaved beta-cryptoxanthin, zeaxanthin, and all-trans-violaxanthin at the 9-10 and 9'-10' positions and 9-cis-violaxanthin at the 9'-10' position. The recombinant CitNCED2 and CitNCED3 proteins cleaved 9-cis-violaxanthin at the 11-12 position to form xanthoxin, a precursor of abscisic acid (ABA). The gene expression of CitCCD1 increased in the flavedos and juice sacs of the three varieties during maturation. In Satsuma mandarin, the gene expression of CitNCED2 and CitNCED3 increased noticeably, accompanying a massive accumulation of ABA in the flavedo and juice sacs. In Valencia orange, the gene expression of CitNCED3 increased with a slight elevation of the ABA level in the flavedo, whereas neither the gene expression of CitNCED2 nor the ABA level increased noticeably in the juice sacs. In Lisbon lemon, the gene expression of CitNCED2 increased remarkably, accompanying increases in the ABA level in the flavedo and juice sacs. These results suggest that, in the juice sacs, the efficient cleavage reaction for ABA synthesis reduces the 9-cis-violaxanthin level in Satsuma mandarin and Lisbon lemon, whereas the low cleavage reaction maintains the predominant 9-cis-violaxanthin accumulation in Valencia orange.

BACKGROUND: Many studies have reported that the consumption of alcohol induces the generation of free radicals. Moreover, recent studies suggest that serum gamma-glutamyltransferase (γ-GTP) within its normal range might be an early marker of oxidative stress. In this study, we tested the hypothesis that serum antioxidant carotenoids would be inversely associated with serum γ-GTP in alcohol drinkers within normal liver function.METHODS: A total of 266 Japanese men who had received health examination in 2003 participated in the study. The associations of serum γ-GTP and serum-carotenoid concentrations stratified by alcohol intake levels were evaluated cross-sectionally. The participants were divided into three groups according to their ethanol intake level (non-drinker, less than 1 g/day; light drinker, 1-25 g/day; and moderate and heavy drinkers, 25+ g/day). The multivariate-adjusted geometric means of the serum γ-GTP concentrations in each tertile of the serum-carotenoid concentrations were calculated after adjustment for ethanol intake, age, body mass index, total cholesterol, triacylglycerols, current tobacco use, and habitual exercise.RESULTS: The serum γ-GTP concentrations were significantly high in accordance with the ethanol intake level. In moderate and heavy drinkers, the multivariate-adjusted geometric means of serum γ-GTP concentrations were significantly low in accordance with the tertiles of the serum lycopene, α-carotene, β-carotene, and β-cryptoxanthin concentrations.CONCLUSIONS: The serum antioxidant carotenoids were inversely associated with alcohol-induced increases of serum γ-GTP in moderate and heavy drinkers within normal liver function.J Epidemiol 2005; 15: 180-186.

The relationship between sugars and ethylene was investigated in harvested broccoli ( Brassica oleracea L. var. italica ) florets. Broccoli florets senesced rapidly after harvest at ambient temperatures, accompanied by the yellowing of sepals, ethylene production and degradation of sugars. In harvested broccoli, continuous uptake of a 10% sucrose (Suc) solution through the cut surface of the stem enhanced ethylene production in florets with simultaneous increases in 1-aminocyclopropane-1-carboxylic acid (ACC) synthase (ACS) activity and ACC content. In spite of the increased rate of ethylene production, the yellowing of sepals and the expression of ACC oxidase (ACO) gene were suppressed by the Suc feeding. When florets were immersed in a 50 mM glucose (Glc) or Suc solution for 24 h, ethylene production was increased by both treatments, and ACS activity and ACC content were the highest in Glc-treated samples. The expression of the ACO gene was enhanced in florets exposed to ethylene (82 μl l −1 ). However, the increases in ethylene production and ACO mRNA accumulation observed in the samples treated with ethylene were suppressed when broccoli plants were treated with ethylene and Suc together. These results indicate that Suc and Glc can affect the rate of ethylene biosynthesis and that high levels of sugars decrease ethylene sensitivity in harvested broccoli florets.

To obtain the quantitative and qualitative data available for estimating the intake of carotenoids from fruits in Japan, carotenoids were analyzed with reversed phase high-performance liquid chromatography (HPLC). Ten carotenoids were examined in 75 raw fruits and 15 processed fruits, all of which were harvested or purchased in Japan. Phytoene was detected in 58 of 90 fruit samples ; ζ-carotene, in 50 of 90 ; lycopene, in 13 of 90 ; α-carotene, in 18 of 90 ; lutein, in 56 of 90 ; β-carotene, in 80 of 90 ; β-cryptoxanthin, in 68 of 90 ; zeaxanthin, in 58 of 90 ; all-trans-violaxanthin, in 55 of 90 ; and 9-cis-violaxanthin, in 47 of 90 samples. Citrus fruits of the mandarin type (Satsuma mandarin and its hybrids, such as tangor) were rich in β-cryptoxanthin, β-carotene, all-trans-violaxanthin, and 9-cis-violaxanthin ; ‘Star ruby’ grapefruit in lycopene ; loquat, Japanese persimmon, and peach in β-cryptoxanthin, β-carotene, all-trans-violaxanthin, and 9-cis-violaxanthin ; mango in β-carotene, all-trans-violaxanthin, and 9-cis-violaxanthin ; acerolas in phytoene ; passion fruits in ζ-carotene. Carotenoid levels in common fruits, such as apple, grape, lemon, pear, strawberry, kiwifruit, cherry, pineapple, and banana, were low.

The ascorbate content declined rapidly in broccoli (Brassica oleracea L. var. italica) florets, but not in the stem tissue, during post-harvest senescence. Ascorbate peroxidase (APX), ascorbate oxidase (AO), l-galactono-1,4-lactone dehydrogenase (GLDH), monodehydroascorbate reductase (MDAR), dehydroascorbate reductase (DHAR), and glutathione reductase (GR) were investigated in gene expression after harvest in both florets and the stem tissue of broccoli. Cytosolic gene expressions (BO-APX 1, BO-APX 2, BO-AO, BO-MDAR 2, and BO-GR) were stimulated actively in broccoli florets after harvest. By contrast, it was observed that mRNA levels of chloroplastic APX, BO-sAPX and BO-tbAPX, had decreased by 12 h after harvest in broccoli florets, suggesting that the active oxygen species (AOS) scavenging system in chloroplasts was largely abolished in florets during the early hours of the post-harvest period. In addition, gene expressions in GLDH and other chloroplastic enzymes such as BO-MDAR 1 and BO-DHAR decreased rapidly within 24 h after harvest. Ethylene treatment had no effect on the ascorbate level and the expression of all genes investigated. The expressions of BO-GLDH and chloroplastic genes (BO-sAPX, BO-tbAPX, BO-MDAR 1, and BO-DHAR) mRNA were suppressed by treatment with methyl jasmonate (MJ) and abscisic acid (ABA) and were accompanied by the acceleration of ascorbate degradation. These data suggest that ascorbate metabolism tends to be inactivated in chloroplasts by transcriptional regulation, but not in the cytosol, when ascorbate decreases under stress conditions.

Two distinct clones having high nucleotide identity to the sequences encoding ascorbate peroxidase (APX) were isolated from broccoli (Brassica oleracea L. var. italica). Deduced amino acid sequences of both cDNAs, BO-APX 1 (accession number AB078599) and BO-APX 2 (accession number AB078600), shared identity of 92.8% and there was more than 80% identity between BO-APXs and other plant cytosolic APXs at the protein level. Gene expression and protein levels of BO-APX 1 and BO-APX 2 were investigated in various parts of broccoli after harvest. Transcript levels of BO-APX 2 gradually increased in florets, while those of BO-APX 1 decreased in florets after harvest. BO-APX 1 and BO-APX 2 were expressed in Escherichiacoli as a fusion protein with glutathione S-transferase (GST) and purified to homogeneity by glutathione sepharose 4B column chromatography. Both proteins of BO-APX 1 and BO-APX 2 appeared as a single major band on SDS-PAGE corresponding to a mass of 25 kDa and reacted with polyclonal antibodies raised against recombinant BO-APX 1. Both enzymes showed high specificities for ascorbate and hydrogen peroxide. The km values of recombinant BO-APX1 and BO-APX 2 for ascorbate were 395 and 526 μM and those for hydrogen peroxide were 15 and 7 μM, respectively. The role of APX was discussed in relation to ascorbate breakdown in broccoli florets during senescence.

Broccoli ( Brassica oleracea , var. italica ) florets (flower buds) senesced rapidly after harvest at ambient temperatures. ACC (1-aminocyclopropane-1-carboxylic acid) synthase (ACS) was induced rapidly in the cut surface of stem tissue in the first 2 mm thick and the basal portions of curds soon after harvest, leading to an accumulation of ACC. The abundance of one ACS mRNA (BO-ACS1) increased in the first layer, the inner layer of stem (2–4 mm, second layer), and the basal portion of curds, while the transcripts for another (BO-ACS2) increased only in the first layer. Conjugated ACC (malonyl ACC, MACC) levels increased in all portions as senescence progressed. In florets, ACC synthase activity and BO-ACS1 transcripts were detected with no significant changes observed during senescence. ACC levels in florets stayed low throughout the experimental period, whereas MACC levels were much higher than those of ACC. The marked rise in ACC oxidase (ACO) activity in florets was detected almost in parallel with a significant increase in ethylene production. The abundance of ACO transcripts (BO-ACO1 and BO-ACO2) increased concurrently with the rise in ACC oxidase activity. These findings suggest that ACC and ethylene synthesized in the stem in response to wounding may have involved the enhanced activity of ACC oxidase and increased abundance of its transcripts in florets.

Dihydrochalcone derivative, 3',5'-di-C-beta-glucopyranosylphloretin (1), is present in the genus Fortunella, (F. crassifolia, F. japonica, F. margarita, F. polyandra and F. hindsii). These species accumulate a large quantity of 1 in their fruits (peel, 6.5-15.2 mg/g in dry wt; juice sac, 1.5-10.5 mg/g) and in their leaves (21.3-60.2 mg/g). Twenty-seven Tanaka's Citrus species examined lack 1, but C. madurensis and C. halimii contain 1 in large quantities in their peels (25.1 and 33.6 mg/g) and juice sacs (4.1 and 4.2 mg/g). Poncirus species do not contain 1. Fortunella-citrus hybrids, the Orangequat [C. unshiuxF. crassifolia], the Thomasville citrangequat [Fortunella sp.x(C. sinensisxPoncirus trifoliata)], and seven hybrid progenies [F. margaritaxC. junos], contain large amounts of 1 in their peels (17.0-7.9 mg/g) and juice sacs (2.0-9.9 mg/g). These facts suggest that accumulation of 1 is a generic trait of the genus Fortunella and that the inheritance of the trait among the intergeneric hybrids is controlled by a dominant allele. Thus C. madurensis and C. halimii are thought to originate from natural hybrids between the genera Citrus and Fortunella. Phloridzin, which has the same aglycon as 1, was not detected in the citrus plants examined.

A cDNA clone encoding a protein homologue to phytoene synthase (PSY, CitPSY1) was isolated from Satsuma mandarin (Citrus unshiu Marc.) fruit. Expression analysis revealed the presence of the CitPSY1 transcript in flowers and leaves, with a higher level detected in the mature than in the young leaves. In fruit, the transcript was present in a low quantity in the flavedo (peel) and juice sacs/segment epidermis (edible part) at an early developmental stage. Later, it began to accumulate towards fruit maturation in both parts. These fluctuations were in accordance with carotenoid accumulation and chlorophyll disappearance. We suggest that CitPSY1 plays an important role in carotenoid accumulation. We also propose a possible pathway in carotenoid biosynthesis that is based on the fluctuation of the characteristic carotenoid composition during Satsuma mandarin development.

A cDNA clone encoding phytoene desaturase (PDS) was isolated from citrus (Citrus unshiu Marc.). The transcript of the isolated PDS (CitPDS1) was not detected by conventional RNA gel-blot analysis; instead, it was detected by a sensitive reverse transcription-PCR (RT-PCR). The CitPDS1 transcript in the juice sacs/segment epidermis (edible part) was at a low level in the young fruit, and it increased toward maturation like citrus phytoene synthase (CitPSY1). In the peel, in contrast to CitPSY1, the transcript of which was induced toward maturation, the level of the CitPDS1 transcript remained constant after an increase in July, indicating non-coordinate regulation of CitPDS1 and CitPSY1 in the peel.

Broccoli (Brassica oleracea L. var. italica) florets senesced rapidly after harvest at ambient room temperatures. Whole plants of broccoli were harvested in the field and brought to the laboratory, then the stem was excised from the root. Ethylene production in the first cross-sectional layer (2 mm thick) initially increased markedly from a trace amount at 0 hr to a peak at 12 hr, then decreased gradually. The rate of ethylene production in florets increased gradually after harvest to reach a maximum at 24 hr. A high amount of ascorbate in florets at harvest, just after separation from the stem, rapidly declined to a low level during senescence. However, in the stem tissue including the cut surface, the ascorbate level which was much less than that of florets, remained almost unchanged during the experimental period. Ascorbate peroxidase (APX), which may be responsible for the first step in ascorbate oxidation retained high activity in broccoli florets after harvest, then decreased slightly during senescence. These results suggest that some additional factors (components) besides high APX activity could be involved in the rapid breakdown of ascorbate.

The rate of ethylene production in sweet potato ( Ipomoea batatas Lam) root tissue greatly increased in response to infection by black rot fungus ( Ceratocystis fimbriata Ell. & Halst.). Ethylene evolved rapidly in the first layer of root cells (0–0.5 mm) 24 h after inoculation with the endoconidia of the fungus. In this layer, the host-parasite interactions were very strong. Ethylene production rate reached a peak (up to 300 nl g −1 h −1 ) 24 h after inoculation, and then declined to a low level. This was followed by an increase in ethylene production in the second layer of cells (0.5–1.0 mm) following continuing invasion by the fungus. Incorporation of l -[ 14 C(U)]methionine into ethylene 24 h after inoculation occurred at a lower rate in the first cell layer than in the second, where the ethylene production rate was about 15-fold less. When ACC (1-aminocyclopropane-1-carboxylic acid) was supplied externally, the rate of ethylene production in the first layer was not enhanced, and neither was [ 14 C]methionine incorporation into ethylene diluted. The activity of ACC oxidase extracted from the first layer was extremely low (less than 10 nl g −1 h −1 ) when compared with the ethylene production rate, although ACC oxidase protein was weakly detected by Western blot analysis in the extract from the infected tissue using antibodies raised against ACC oxidase purified from recombinant Escherichia coli . These results indicate that the predominant ethylene generated in the site adjacent to the invaded region of sweet potato root tissue may originate from a pathway independent of ACC. However, it is possible that the methionine-ACC pathway may operate in tissue internal to the above active site.

A cDNA clone encoding a protein (CuCOR19), the sequence of which is similar to Poncirus COR19, of the dehydrin family was isolated from the epicarp of Citrus unshiu. The molecular mass of the predicted protein was 18,980 daltons. CuCOR19 was highly hydrophilic and contained three repeating elements including Lys-rich motifs. The gene expression in leaves increased by cold stress.

A cDNA (CitPAP) homologous to a gene encoding for Cucumis sativus carotenoid-associated protein (CHRC) has been isolated from satsuma mandarin (Citrus unshiu Marc.). Unlike ChrC whose expression was limited only in mature fruits (containing chromoplasts), CitPAP transcripts were detected in all the tissues examined including fruits, flowers and leaves. In this respect, CitPAP was rather close to a gene encoding for pepper plastid-lipid-associated protein (PAP), which exhibits ubiquitous expression in bell pepper organs containing chloroplasts or chromoplasts. CitPAP, however, differed from PAP in the magnitude and pattern of RNA accumulation. These results might indicate a novel function of CitPAP.

To use recombinant 1-aminocyclopropane-1-carboxylate (ACC) oxidase for research on ethylene biosynthesis, the biochemistry of both recombinant and endogenous ACC oxidase from kiwifruit was compared. When induced by the addition of isopropyl-β- d -thiogalactopyranoside (IPTG) to Escherichia coli (E. coli) cells transformed with cDNA AD-ACO1 using the pGEX-4T-1 vector, ACC oxidase identical to that from kiwifruit was expressed as a polypeptide of 37 kDa. Apparent Km values for ACC for both recombinant and endogenous ACC oxidase were 41 and 16 μM, respectively. Both forms of ACC oxidase exhibited absolute requirements for ferrous iron, ascorbate and bicarbonate for maximum activity The activities of both enzymes were inhibited by ethylenediaminetetraacetic acid (EDTA), 4,5-dihydroxy-1,3-benzenedisulfonic acid (Tiron), o-phenanthroline (PA), α-aminoisobutyric acid (AIB), and p-chloromercuriphenylsulfonic acid (PCMPS). Addition of dithiothreitol (DTT) stimulated activity of both ACC oxidases. The results indicate that the recombinant ACC oxidase was similar biochemically to the endogenous kiwifruit enzyme. Western blot analysis using antibody raised against purified transformed ACC oxidase protein showed differential expression of endogenous ACC oxidase protein in kiwifruit during ripening. Expression may start in the columella region and then increase in surrounding tissues with progressive stages of ripening.

Parthenocarpic kiwifruit Actinidia deliciosa was produced and induction of ethylene synthesis in response to ethylene treatment studied. At anthesis, flowers were treated with N -(2-chloro-4-pyridyl)- N ′-phenylurea (CPPU). CPPU-treated parthenocarpic fruit and pollinated normal fruit were harvested in early November, and then treated with 1000 μ l l −1 ethylene at 20°C for 24 h. Ethylene production following ethylene treatment was reduced and delayed in the parthenocarpic fruit. To investigate the mechanism of this reduction in ethylene synthesis, 1-aminocyclopropane-1-carboxylic acid (ACC) concentrations, and gene expression of ACC synthase (using a cDNA probe, AD-ACS1) and of ACC oxidase (with a cDNA probe, AD-ACO1) were analyzed. ACC concentrations and levels in ACC synthase transcripts increased in the pollinated fruit 2 days after the 24 h ethylene treatment, but not in the parthenocarpic fruit. Accumulation of ACC oxidase transcripts was stimulated immediately by the 24 h ethylene treatment in both pollinated and parthenocarpic fruit. These results suggest that suppression of ethylene synthesis in parthenocarpic fruit resulted mainly from suppressed expression of ACC synthase.

Large varietal differences in ethylene production in Chinese kiwifruits were found, especially between 'Kui mi' and 'Hong xin', based on the sensitivity and responsiveness of the cultivars to exogenous ethylene, which could induce autocatalytic ethylene production in fruit. The difference in the potential to induce ethylene production is attributed to the greater gene expression of ACC synthase in 'Kui mi' than in 'Hong xin'. In contrast, no distinct differences were found in the transcript levels of ACC oxidase gene between these cultivars. 'Kui mi' having a high rate of ethylene production also had a high ACC content compared with 'Hong xin'. When endogenous ethylene production reached a peak at 96 hr after 24 hr-ethylene treatment, the transcript levels of ACC synthase gene were most prominent. However, the transcript level of ACC oxidase gene was clearly detected in all samples of 0 hr and more after a 24 hr-ethylene treatment. These results suggest that a key enzyme which controls the rate of endogenous ethylene production in kiwifruit is ACC synthase, not ACC oxidase.

Methods for extracting total RNA from various tissues of satsuma mandarin and kiwifruit were examined. Satisfactory yields in total RNA were obtained from tissues with the exception of the albedo tissue of satsuma mandarin, by using a modification of the conventional extraction method devised for fruit tissues by Lopez-Gomez and Gomez-Lim (1992). In the albedo tissue, the polysaccharides may interfere with RNA extraction but further modification of the extraction method improved its yield. The modification involved repeated back extraction, chloroform/isoamyl alcohol extraction, and increasing the volume of the aqueous phase before precipitating RNA with LiCl (3 M final concentration). From the total RNA, poly (A)+RNA was purified using an oligo (dT) -cellulose column. The poly (A)+RNA could be successfully used for Reverse Transcription-Polymerase Chain Reaction (RT-PCR) and the construction of a cDNA library. This modified protocol is applicable to other fruit tissues rich in polysaccharides.

Ethylene is involved in the premature senescence of broccoli after harvest. In an effort to understand the regulation of ethylene responses, we isolated three partial cDNAs from broccoli, BO-ETR1, BO-ERS, and BO-ETR2 as putative ethylene receptors. BO-ETR2 is a novel cDNA and it shared 77%, 68%, and 58% nucleotide identities with Arabidopsis ETR2 and tomato LeETR4, and BO-ETR1, respectively, in the corresponding regions of 479 bp long. Northern-blot analysis revealed that BO-ETR1 and BO-ERS mRNA were expressed in all examined parts of stem tissue and florets, but the transcription levels were higher in the stem tissue than in florets. During the broccoli senescence no significant change occurred in the expression of BO-ETR1 gene in either the stem or floret tissue. BO-ERS transcripts increased during the first 12 hr after harvest in the first layer of stem cutting, but decreased in the basal portion of curds. BO-ETR2 mRNA, which was maximum at harvest (0 hr), was detected only in the stem tissue. A significant reduction of BO-ETR2 transcripts was observed 36 hr after harvest. The results indicate that the gene expression for the above putative ethylene receptors in broccoli is regulated in a tissue specific manner.

Seasonal changes in the physical properties and chemical constituents of fruits during the maturing stage were examined in 5 strains of very early ripening mutants of Satsuma mandarin (Citrus unshiu Marc. var praecox Tanaka) with special reference to the limonoid compounds.The fruit size showed no significance during harvests performed 5 times successively. The proportion of peel of 'Hashimoto wase' was higher than other strains and peel-puffing was occurred. The Brix value increased in almost all the strains during fruit maturation and the sucrose contents mainly increased among the sugar constituents. Organic acid contents decreased, in particular citric acid as in the case of Wase satsuma mandarin. Both contents of limonin and nomilin were reduced with fruit maturation, but the rate of decrease of nomilin was faster than that of limonin. In the fruits harvested late, juice processing was optimal in terms of chemical components except for 'Hashimoto wase'.Since the correlation coefficients between the Brix value, citric acid or rind color a-value and limonoid compounds were very high, bitterness due to the presence of limonoids in the juice products could be predicted from several parameters. Based on the principal component analysis of the juice characteristics, 'Hashimoto wase' displayed different properties compared with the other strains. Based on this statistical method, it may become possible to develop a new classification of very early ripening mutants of Satsuma mandarin.

The physical and chemical properties of fruits used as raw materials for juice processing were examined among 12 strains in 6 classes of very early ripening mutants of Wase satsuma mandarin (Citrus unshiu Marc. var. praecox Tanaka) with special reference to the limonoid compounds.All the strains used in these experiments completely fulfilled the criteria of the Japanese Agricultural Standard (JAS) for single-strength juice. On the basis of these criteria, the sugar, citric acid, ascorbic acid (vitamin C), amino-nitrogen and ash contents in the very early ripening mutants were suitable for juice processing.However, the contents of limonoid compounds of these strains were much higher than those of Wase and Common satsuma mandarin at the usual harvest time. Such high contents may cause the so called "delayed bitterness" in their juice products after processing with an In-Line juice extractor. Therefore, to alleviate this shortcoming, it is suggested that the fruits should be harvested at the full ripening stage.Although the mutants were classified into 6 maturation types based on the variations of acid contents and coloring time of the fruit, the maturation time could not be estimated on the basis of the contents of limonoid compounds.

Large differences in gene expression of 1-aminocyclopropane-1-carboxylic acid (ACC) synthase, ACC contents, and ACC synthase activitiy were found in different tissues of fruit between members of two kiwifruit species, (cv) Actinidia chinensis ('Kui mi') and A. deliciosa ('Hayward'). ACC synthase mRNA accumulated mostly in the outer pericarp, slightly in the inner pericarp, and in a trace quantity in the columella of 'Hayward' fruit that produces much less ethylene than does 'Kui mi'. However, gene transcripts at significant levels exist in all sections of 'Kui mi' fruit which reflects a large potential to produce ethylene. ACC levels and ACC synthase activities were much higher in all the tissues of 'Kui mi' fruit than in 'Hayward' fruit, which indicates that these enzymes are the primary cause for the varietal difference in the ability to produce ethylene in kiwifruit.