Cytologic And Metabolic Studies Of Engineered Carotenoid Accumulation In Citrus

Carotenoids are plant indispensable secondary metabolites, involved in a series of biological processes, such as photosynthesis, antioxidation, hormone biosynthesis, and attractants for pollinators. Carotenoids are also essential health-protecting compounds for supplying provitamin A, suppressing the development of several chronic diseases, and being involved in human’s immunity, embryonic development, and reproduction. Plant is one of the major organisms containing carotenogenesis, and is also the most important carotenoid source for human life. To exert their effective usage, it is essential to understand the regulation mechanism of plant carotenoid metabolism.Citrus exhibits extensive diversity of carotenoid patterns, and is the better material for plant carotenoid metabolism study. In this study, by creating various genetic resources, including transgenic embryogenic calli, transgenic plants, and Valencia sweet orange DH lines, we studied the regulation mechanism of carotenoid metabolism, correlation between carotenoid metabolism and other biological processes, and allelic diversity of two key carotenogenic genes LCYB1and LCYE in sweet orange. The main results were as followed:1. Regulating carotenoid accumulation through overexpressing CrtB and DSM2in citrus embryogenic calli(1) We engineered embryogenic calli from Marsh grapefruit(Citrus paradise Macf.), Star Ruby grapefruit(Citrus paradise Macf.), Cara Cara navel orange [Citrus sinensis (L.) Osb.], and Sunburst mandarin [Citrus reticulata Blanco×(Citrus paradisi Macf.×Citrus Reticulate)] as Engineered Cell Models (ECMs) by overexpressing CrtB (a bacterial phytoene synthase gene). In the ECMs, the levels of total carotenoids markedly increased, and carotenoid patterns showed diversity depending on the genotypes.30free-type carotenoids were detected in the ECMs, including all of types in wild types and some new ones, such as lycopene.(2) Especially in the ECMs from dark-grown culture, there emerged a favored β,β-pathway characterized by a striking accumulation of β-carotene, which was dramatically unique from those in the wild-type calli. Based on the data in this study, a model was established to explain the favoring of β,β-pathway in the ECMs. This model suggests that LCYE (lycopene ε cyclase) plays a bottleneck role in the presence of abundant lycopene substrates leading to an altered carotenoid composition.(3) It was noted that the ECMs under irradiation showed a marked decrease in β-carotene and phytoene levels, while lutein kept stable. Most carotenoids were also reduced in37℃grown ECM line, but zeaxanthin level increased significantly. In addition, ECM exhibited unstable CrtB transcription and carotenoid accumulation, which is associated with epigenetic regulation.(4) Ripe flavedos (the colored outer layer of citrus fruits) from four consistent genotypes were offered as a comparative system to the ECMs. The comparative analysis showed ripe flavedos contained chiefly esterified violaxanthin and cryptoxanthin, or lycopene. Unlike flavedos, the ECMs did not form chromoplast, instead they sequestered most carotenoids in the amyloplasts in crystal form. Conglomerates containing abundant carotenoids were observed in the vacuoles of ECMs, suggesting an underlying catabolism mechanism that governs carotenoids.(5) Transcriptional analysis revealed that most of isoprenoid and carotenoid genes exhibited up-regualted expression in flavedos, especially HYB (gene encoding β-carotene hydroxylase) showed a markedly flavedo-specific expression, but was suppressed in the calli. Furthermore, coexpression of CrtB and DSM2(a HYD gene from rice) in the ECMs confirmed that HYD predominantly mediated the preferred carotenoid patterns between the ECMs and flavedos, and also unraveled that the carotenoid crystals in the ECMs were majorly composed of P-carotene. In addition, transcript perturbation of endogenous carotenogenic genes was observed in ECMs, especially in the transgenic callus lines of Star Ruby grapefruit.2. Engineered carotenoid accumulation affects plastid development and metabolsim(1) Transgenic callus lines with overexpressive CrtB gene (ECMs) accumulated lower levels of starch and higher levels of soluble sugars relative to wild types, showing the effect of carotenoid accumulation on carbohydrate metabolism, and also suggesting a potential chromoplast development in the ECMs. Though sucrose-free culture, we found ECMs indeed had the chromoplast development program, while it was suppressed by the predominant starch accumulation under normal culture.(2) Global transcriptional diversities between ECMs and wild types of four genotypes were investigated through citrus microarray analysis. The results showed similar diversity pattern existed in four groups, which was characterized by a marked up-regulation of abundant stress or redox response genes, such as POD (gene encoding peroxidase), GST (gene encoding glutathione S transferase), HSP (gene encoding heat shock protein) and the genes involved in phenylpropanoid metabolism. In addition, some anthocyanin genes were down-regulated in the ECMs, which suggested that carotenoid accumulation could negatively regulated anthocyanin biosynthesis. We discovered a-amylase gene up-regulated in the ECMs, which was probably related to the alteration of starch content in the ECMs.(3) ABA levels in the ECMs showed little correlation with stress and redox response. Furthermore, reactive oxidative species (ROS) were investigated, and the result showed that carotenoid accumulation could alter cellular redox status. ECMs contained lower O2-(superoxide radical) levels compared to wild types, while H2O2(hydrogen peroxide) levels were higher in the ECMs. Phenotypes of the ECM treated by concentrated sucrose and reductives suggested that O2-could be involved in the degradation of carotenoids in the ECMs. In addition, this study showed that redox status potentially affected starch metabolism in citrus callus.(4) To confirm that carotenoid accumulation could negatively regulate anthocyanin biosynthesis, an apple ECM was constructed through overexpressing CrtB gene in apple callus with anthocyanin accumulation. This ECM yielded more carotenoids than wild type, but anthocyanin biosynthesis was suppressed. Norflurazon, an inhibitor of plant carotenoid biosynthesis could partially resume anthocyanin accumulation in the apple ECM. Further transcriptional analysis showed that anthocyanin biosynthetic genes were down-regulated in this ECM.3. Transformation with CrtB gene in precocious trifoliate orange and Hongkong kumquat, and study of carotenoid accumulative characters in transgenic plants(1) Through CrtB transformation of early flowering citrus, we obtained one transgenic precocious trifoliate orange [Poncirus trifoliate (L.) Raf] line and two transgenic Hongkong kumquat (Fortunella hindsii Swingle) lines. Regenerated transgenic shoots of precocious trifoliate orange showed orange, suggesting a high carotenoid accumulation. Whereas, most of orange regenerated shoots fail to become green, and could not develop further. One regenerated transgenic precocious trifoliate orange shoot with orange to green transition was further cultured, it showed dwarf phenotype.(2) Green tissues of transgenic Hongkong kumquats had no orange phenotype, while their petals, senescent leafstalks, seeds, and roots exhibited orange, suggesting high accumulation of carotenoids. Roots of wild-type seedlings could become green under light-grown culture, while roots of transgenic seedlings kept orange. In addition, the ripe fruits of transgenic Hongkong kumquat1(TSJ-1) showed diverse color:one represented orange similar to wild type, another one represented yellow; the ripe fruits of TSJ-2exhibited a little lighter color compared with those of wild type.(3) Plastid inspection revealed that, in the cells of senescent leafstalks and light-grown roots of wild type, chloroplasts were main plastid type, while chromoplasts were observed in transgenic cells; abundant amyloplasts existed chiefly in the cells of wild-type petals, dark-grown roots and embryoids, but chromoplasts appeared predominantly in transgenic cells. All these microscopic data confirmed that carotenoid accumulation confers chromoplast development in transgenic Hongkong kumquat. In addition, the yellow fruits of TSJ-1and mature fruits of TSJ-2both contained abundant electron-dense plastoglobules, but showed lower amount of chromoplast and plastoglobules than wild-type fruits.(4) Compared to wild types, all three-year-old leaves of two transgenic Hongkong kumquats almost abscised, suggesting an early senescence symptom. Leaves of transgenic Hongkong kumquats contained higher ABA contants, which could induce the early senescence in transgenic Hongkong kumquats.4. Recovery of Rohde Red sweet orange DH line for studying allelic diversity of two carotenogenic genes LCYB1and LCYE in sweet orangeTwo callus lines of Valencia sweet orange cv. Rohde Red [Citrus sinensis (L.) Osbeck] were recovered through anther culture in this study. Ploidy investigation and SSR analysis revealed two callus lines were doubled haploids (DHs). DHs provided support for the allelic diversity of two carotenogenic genes, LCYE and LCYB, in sweet orange.In summary, this study shows a valuble approach to comprehension of the nature of carotenoid metabolism in citrus and other plants, and supplies some important basic data with regard to color improvement of citrus fruit, and also provides an essential material for citrus genome sequence.