Novel expression patterns of carotenoid pathway-related genes in citrus leaves and maturing fruits

Carotenoids are abundant in citrus fruits and vary among cultivars and species. In the present study, high performance liquid chromatography (HPLC) and real-time polymerase chain reaction (PCR) were used to investigate the expression patterns of 23 carotenoid biosynthesis gene family members and their possible relation with carotenoid accumulation in fresh flavedo, juice sacs and leaves of Valencia orange during fruit maturation. Violaxanthin and lutein mainly accumulated in fruit (flavedo and juice sacs) and leaves (young and mature), respectively, accounting for nearly 79 %, 57 %, 53 % and 70 % of corresponding total carotenoids in February. Violaxanthin content quickly began to increase in flavedo in December, but the increase in juice sacs began later in January. In mature leaves, lutein content was three times that in young leaves; α-carotene and β-carotene were also much higher in mature leaves than in flavedo or juice sacs. Generally most of the carotenoid biosynthesis gene members were expressed at higher levels in flavedo than in juice sacs, and the expression of some continued to increase in flavedo during fruit maturation. All CHYB members expressed at high levels and had similar patterns in juice sacs. Interestingly, the capsanthin capsorubin synthase (CCS) members had similar expression levels and patterns in flavedo and juice sacs. Differences in gene expression between leaf and fruit tissues were noted, pointing to some tissue specificity for certain members of the gene families associated with carotenogenesis. The expression patterns of these 23 citrus carotenoid biosynthesis gene members were also compared with their expression patterns in other plants. Taken together, these first-hand expression data will be useful to define the tissue-specific roles of each gene member in accumulation of different carotenoids in citrus leaves and maturing fruits.     

Expression of three isoprenoid biosynthesis genes and their effects on the carotenoid production of the zygomycete Mucor circinelloides

The zygomycete Mucor circinelloides accumulates β-carotene as the main carotenoid compound. In this study, the applicability of some early genes of the general isoprenoid pathway to improve the carotenoid production in this fungus was examined. The isopentenyl pyrophosphate isomerase gene (ipi) was cloned and used together with the genes encoding farnesyl pyrophosphate synthase (isoA) and geranylgeranyl pyrophosphate synthase (carG) in overexpression studies. Transformation experiments showed that the first bottleneck in the pathway, from the aspect of carotenoid production, is the step controlled by the carG gene, but overexpression of the ipi and isoA genes also contributes to the availability of the precursors. Transformations with these isoprenoid genes in combination with a bacterial β-carotene ketolase gene yielded Mucor strains producing canthaxanthin and echinenone.     

木薯品种(系)类胡萝卜素代谢相关基因和蛋白表达水平分析

以白心木薯华南6068、华南9号、紫叶黄心木薯BGM019和粉红木薯Mirasol为材料,探究木薯块根膨大期和成熟期与类胡萝卜素代谢通路相关的14个基因和4种蛋白质表达水平变化。用HPLC检测块根β-胡萝卜素含量的变化,分别用qRT-PCR和Western blot方法对类胡萝卜素代谢通路相关基因和蛋白酶的表达水平进行分析。以华南6068为对照,研究结果表明:华南9号和紫叶黄心木薯BGM019成熟期中的类胡萝卜素合成途径关键基因PSY2、LCYB基因显著高于膨大期,而降解相关的关键基因CCD1、NCED3在成熟期的表达量显著低于膨大期(P0.05)。粉红木薯Mirasol成熟期中PSY2、LCYB的显著下调与CCD1、NCED3的显著上调(P0.05)是造成β-胡萝卜素含量差异的原因之一。通过分析不同木薯品种(系)在膨大期和成熟期块根类胡萝卜素代谢途径相关基因的表达水平,有助于解析β-胡萝卜素积累的分子机理。此外,Western blot结果显示抗坏血酸过氧化物酶、谷胱甘肽还原酶、超氧化物歧化酶和HSP70虽然和块根类胡萝卜素代谢途径没有直接关联,但它们在木薯膨大期和成熟期块根表达水平有显著差异(P0.05)。     

Ethylene regulation of carotenoid accumulation and carotenogenic gene expression in colour-contrasted apricot varieties (Prunus armeniaca)

In order to elucidate the regulation mechanisms of carotenoidbiosynthesis in apricot fruit (Prunus armeniaca), carotenoidcontent and carotenogenic gene expression were analysed as afunction of ethylene production in two colour-contrasted apricotvarieties. Fruits from Goldrich (GO) were orange, while Moniqui(MO) fruits were white. Biochemical analysis showed that GOaccumulated precursors of the uncoloured carotenoids, phytoeneand phytofluene, and the coloured carotenoid, ß-carotene,while Moniqui (MO) fruits only accumulated phytoene and phytofluenebut no ß-carotene. Physiological analysis showed thatethylene production was clearly weaker in GO than in MO. Carotenogenicgene expression (Psy-1, Pds, and Zds) and carotenoid accumulationwere measured with respect to ethylene production which is initiatedin mature green fruits at the onset of the climacteric stageor following exo-ethylene or ethylene-receptor inhibitor (1-MCP)treatments. Results showed (i) systematically stronger expressionof carotenogenic genes in white than in orange fruits, evenfor the Zds gene involved in ß-carotene synthesisthat is undetectable in MO fruits, (ii) ethylene-induction ofPsy-1 and Pds gene expression and the corresponding productaccumulation, (iii) Zds gene expression and ß-caroteneproduction independent of ethylene. The different results obtainedat physiological, biochemical, and molecular levels revealedthe complex regulation of carotenoid biosynthesis in apricotsand led to suggestions regarding some possible ways to regulateit. Key words: Apricot, carotenoid, ethylene, fruit, 1-MCP, Prunus armeniaca, ripening-related genes     

Functional assignment of Erwinia herbicola Eho10 carotenoid genes expressed in Escherichia coli

Erwinia herbicola is a nonphotosynthetic bacterium that is yellow pigmented due to the presence of carotenoids. When the Erwinia carotenoid biosynthetic genes are expressed in Escherichia coli, this bacterium also displays a yellow phenotype. The DNA sequence of the plasmid pPL376, carrying the entire Erwinia carotenoid gene cluster, has been found to contain 12 open reading frames (ORFs). Six of the ORFs have been identified as carotenoid biosynthesis genes that code for all the enzymes required for conversion of farnesyl pyrophosphate (FPP) to zeaxanthin diglucoside via geranylgeranyl pyrophosphate, phytoene, lycopene, β-carotene, and zeaxanthin. These enzymatic steps were assigned after disruption of each ORF by a specific mutation and analysis of the accumulated intermediates. Carotenoid intermediates were identified by the absorption spectra of the colored components and by high pressure liquid chromatographic analysis. The six carotenoid genes are arranged in at least two operons. The gene coding for β-carotene hydroxylase is transcribed in the opposite direction from that of the other carotenoid genes and overlaps with the gene for phytoene synthase.     

Rice carotenoid β-ring hydroxylase CYP97A4 is involved in lutein biosynthesis

Lutein is the most abundant plant carotenoid and plays essential roles in photosystem assembly and stabilization, as well as protection against photostress. To date, only a few lutein biosynthesis genes have been identified in crop plants. In this study, the rice Cyt P450 gene CYP97A4 encoding a carotenoid β-ring hydroxylase was shown to be involved in lutein biosynthesis. The results revealed that CYP97A4 was preferentially expressed in leaf compared with spikelet, sheath, stalk and root, and encoded a protein localized at the subcellular level to the chloroplasts. Compared with the wild type, the three allelic mutants of CYP97A4 displayed lutein reductions of 12-24% with substantially increased α-carotene, while Chl a/b levels were unaltered. The increased α-carotene in the mutants led to greater sensitivity under high light stress. Similarly, reactive oxygen species (ROS) imaging of leaves treated with intense light showed that the mutants generally accumulated greater levels of ROS compared with wild-type plants, which probably caused detrimental effects to the plant photosystem. In conclusion, this study demonstrated the important role of CYP97A4 in α-carotene hydroxylation in rice, and knock-out of the gene reduced lutein and increased α-carotene, contributing to sensitivity to intense light.