Blue LED light induces regreening in the flavedo of Valencia orange in vitro.

In the present study, the effects of blue LED light on the regreening of citrus fruit were investigated in an in vitro system of Valencia orange flavedos. The results showed that blue LED light irradiation induced regreening in the flavedos. After four-week culture in vitro, the flavedos exhibited obviously green color in the blue LED light treatment, while the flavedos in the control were still in orange color. During the regreening process, the blue LED light treatment induced chlorophyll accumulation, and substantially altered the carotenoid composition in the flavedos. Compared with the control, the content of 9-cis-violaxanthin was decreased, while the contents of lutein, ß-carotene, and all-trans-violaxanthin were increased by blue LED light. In addition, gene expression results showed that the up-regulation of CitLCYe and down-regulation of CitLCYb2 by blue LED light led to a shift from ß,ß-branch to ß,ε-branch of the carotenoid biosynthetic pathway.

Expression and functional analysis of the nobiletin biosynthesis-related gene CitOMT in citrus fruit.

Nobiletin, a polymethoxy flavone (PMF), is specific to citrus and has been reported to exhibit important health-supporting properties. Nobiletin has six methoxy groups at the 3',4',5,6,7,8-positions, which are catalyzed by O-methyltransferases (OMTs). To date, researches on OMTs in citrus fruit are still limited. In the present study, a novel OMT gene (CitOMT) was isolated from two citrus varieties Satsuma mandarin (Citrus unshiu Marc.) and Ponkan mandarin (Citrus reticulata Blanco), and its function was characterized in vitro. The results showed that the expression of CitOMT in the flavedo of Ponkan mandarin was much higher than that of Satsuma mandarin during maturation, which was consistent with the higher accumulation of nobiletin in Ponkan mandarin. In addition, functional analysis showed that the recombinant protein of CitOMT had methylation activity to transfer a methyl group to 3'-hydroxy group of flavones in vitro. Because methylation at the 3'-position of flavones is vital for the nobiletin biosynthesis, CitOMT may be a key gene responsible for nobiletin biosynthesis in citrus fruit. The results presented in this study will provide new strategies to enhance nobiletin accumulation and improve the nutritional qualities of citrus fruit.

Accumulation of carotenoids in a novel citrus cultivar 'Seinannohikari' during the fruit maturation.

In the present study, carotenoid metabolism was investigated in the fruits of a novel citrus cultivar, 'Seinannohikari' (Citrus spp.). During the maturation, ß,ß-xanthophylls were accumulated rapidly with ß-cryptoxanthin being the dominant carotenoid compound in the flavedo and juice sacs of 'Seinannohikari'. In the juice sacs of mature fruits, 'Seinannohikari' accumulated high amount of carotenoids, especially ß-cryptoxanthin. The content of ß-cryptoxanthin in the juice sacs of 'Seinannohikari' was approximately 2.5 times of that in 'Miyagawa-wase' (Citrus unshiu), which is one of its parental cultivars, at the mature stage. Gene expression results showed that the massive accumulation of ß-cryptoxanthin might be attributed to the higher expression of carotenoid biosynthetic genes (CitPSY, CitPDS, CitZDS, CitLCYb2, CitHYb, and CitZEP), and lower expression of carotenoid catabolic genes (CitNCED2 and CitNCED3) in the juice sacs of 'Seinannohikari'.

Identification and quantitative analysis of ß-cryptoxanthin and ß-citraurin esters in Satsuma mandarin fruit during the ripening process.

In this study, to investigate the xanthophyll accumulation in citrus fruits, the major fatty acid esters of ß-cryptoxanthin and ß-citraurin were identified, and changes in their contents were investigated in two Satsuma mandarin varieties, 'Miyagawa-wase' and 'Yamashitabeni-wase', during the ripening process. The results showed that ß-cryptoxanthin and ß-citraurin were mainly esterified with lauric acid, myristic acid, and palmitic acid in citrus fruits. During the ripening process, ß-cryptoxanthin laurate, myristate, and palmitate were accumulated gradually in the flavedos and juice sacs of the two varieties. In the flavedo of 'Yamashitabeni-wase', ß-citraurin laurate, myristate, and palmitate were specifically accumulated, and their contents increased rapidly with a peak in November. In addition, functional analyses showed that CitCCD1 and CitCCD4 efficiently cleaved the free ß-cryptoxanthin, but not the ß-cryptoxanthin esters in vitro. The substrate specificity of CitCCDs towards free ß-cryptoxanthin indicated that ß-cryptoxanthin esters might be more stable than free ß-cryptoxanthin in citrus fruits.

Expression and functional analysis of citrus carotene hydroxylases: unravelling the xanthophyll biosynthesis in citrus fruits.

BACKGROUND: Xanthophylls are oxygenated carotenoids and fulfill critical roles in plant growth and development. In plants, two different types of carotene hydroxylases, non-heme di-iron and heme-containing cytochrome P450, were reported to be involved in the biosynthesis of xanthophyll. Citrus fruits accumulate a high amount of xanthophylls, especially ß,ß-xanthophylls. To date, however, the roles of carotene hydroxylases in regulating xanthophyll content and composition have not been elucidated. RESULTS: In the present study, the roles of four carotene hydroxylase genes (CitHYb, CitCYP97A, CitCYP97B, and CitCYP97C) in the biosynthesis of xanthophyll in citrus fruits were investigated. Phylogenetic analysis showed that the four citrus carotene hydroxylases presented in four distinct clusters which have been identified in higher plants. CitHYb was a non-heme di-iron carotene hydroxylase, while CitCYP97A, CitCYP97B, and CitCYP97C were heme-containing cytochrome P450-type carotene hydroxylases. Gene expression results showed that the expression of CitHYb increased in the flavedo and juice sacs during the ripening process, which was well consistent with the accumulation of ß,ß-xanthophyll in citrus fruits. The expression of CitCYP97A and CitCYP97C increased with a peak in November, which might lead to an increase of lutein in the juice sacs during the ripening process. The expression level of CitCYP97B was much lower than that of CitHYb, CitCYP97A, and CitCYP97C in the juice sacs during the ripening process. Functional analysis showed that the CitHYb was able to catalyze the hydroxylation of the ß-rings of ß-carotene and α-carotene in Escherichia coli BL21 (DE3) cells. Meanwhile, when CitHYb was co-expressed with CitCYP97C, α-carotene was hydroxylated on the ß-ring and ε-ring sequentially to produce lutein. CONCLUSIONS: CitHYb was a key gene for ß,ß-xanthophyll biosynthesis in citrus fruits. CitCYP97C functioned as an ε-ring hydroxylase to produce lutein using zeinoxanthin as a substrate. The results will contribute to elucidating xanthophyll biosynthesis in citrus fruits, and provide new strategies to improve the nutritional and commercial qualities of citrus fruits.

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.

Fairy Chemicals, 2-Azahypoxanthine and 2-Aza-8-oxohypoxanthine, Regulate Carotenoid Accumulation in Citrus Juice Sacs in Vitro.

"Fairy chemicals", 2-azahypoxanthine (AHX) and 2-aza-8-oxohypoxanthine (AOH), are two novel plant-growth regulating compounds isolated from a fairy ring forming fungus, Lepista sordida. In the present study, the effects of AHX and AOH on the accumulation of carotenoids and expression of genes related to carotenoid metabolism were investigated in the juice sacs of Satsuma mandarin (Citrus unshiu Marc.) in vitro. The results showed that AHX and AOH regulated carotenoid metabolism in the citrus juice sacs. Carotenoid accumulation was induced by AHX in the second week and by AOH in the fourth week. In the meanwhile, the modification of carotenoid accumulation by the AHX and AOH treatments was highly regulated at the transcriptional level. The results presented herein provide new information on the functions of AHX and AOH in plants and contribute to elucidating the mechanisms by which AHX and AOH stimulate plant growth.

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.

Enzymatic formation of ß-citraurin from ß-cryptoxanthin and Zeaxanthin by carotenoid cleavage dioxygenase4 in the flavedo of citrus fruit.

In this study, the pathway of ß-citraurin biosynthesis, carotenoid contents and the expression of genes related to carotenoid metabolism were investigated in two varieties of Satsuma mandarin (Citrus unshiu), Yamashitabeni-wase, which accumulates ß-citraurin predominantly, and Miyagawa-wase, which does not accumulate ß-citraurin. The results suggested that CitCCD4 (for Carotenoid Cleavage Dioxygenase4) was a key gene contributing to the biosynthesis of ß-citraurin. In the flavedo of Yamashitabeni-wase, the expression of CitCCD4 increased rapidly from September, which was consistent with the accumulation of ß-citraurin. In the flavedo of Miyagawa-wase, the expression of CitCCD4 remained at an extremely low level during the ripening process, which was consistent with the absence of ß-citraurin. Functional analysis showed that the CitCCD4 enzyme exhibited substrate specificity. It cleaved ß-cryptoxanthin and zeaxanthin at the 7,8 or 7',8' position. But other carotenoids tested in this study (lycopene, α-carotene, ß-carotene, all-trans-violaxanthin, and 9-cis-violaxanthin) were not cleaved by the CitCCD4 enzyme. The cleavage of ß-cryptoxanthin and zeaxanthin by CitCCD4 led to the formation of ß-citraurin. Additionally, with ethylene and red light-emitting diode light treatments, the gene expression of CitCCD4 was up-regulated in the flavedo of Yamashitabeni-wase. These increases in the expression of CitCCD4 were consistent with the accumulation of ß-citraurin in the two treatments. These results might provide new strategies to improve the carotenoid contents and compositions of citrus fruits.

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.

Effect of blue and red LED light irradiation on ß-cryptoxanthin accumulation in the flavedo of citrus fruits.

ß-Cryptoxanthin (ß-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 ß-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 ß-cry was induced by red light, while it was not affected by blue light. The accumulation of ß-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.

Transgenic Arabidopsis plants expressing the rice dehydroascorbate reductase gene are resistant to salt stress.

Vitamin C (l-ascorbate) is important for antioxidative and metabolic functions in both plants and humans. Ascorbate itself is oxidized to dehydroascorbate during the process of antioxidation, and dehydroascorbate reductase (DHAR, EC 1.8.5.1) re-reduces the oxidized ascorbate. Therefore, this enzyme is assumed to be critical for ascorbate recycling. Here we show that the expression of rice DHAR in transgenic Arabidopsis thaliana enhanced resistance to salt stress. Salt tolerance was remarkably improved despite slight increases in DHAR activity and total ascorbate. This study provides direct evidence for the importance of DHAR in salt tolerance.