Regulation and activation of phytoene synthase, a key enzyme in carotenoid biosynthesis, during photomorphogenesis

 During photomorphogenesis in higher plants, a coordinated increase occurs in the chlorophyll and carotenoid contents. The carotenoid level is under phytochrome control, as reflected by the light regulation of the mRNA level of phytoene synthase (PSY), the first enzyme in the carotenoid biosynthetic pathway. We investigated PSY protein levels, enzymatic activity and topological localization during photomorphogenesis. The results revealed that PSY protein levels and enzymatic activity increase during de-etiolation and that the enzyme is localized at thylakoid membranes in mature chloroplasts. However, under certain light conditions (e.g., far-red light) the increases in PSY mRNA and protein levels are not accompanied by an increase in enzymatic activity. Under those conditions, PSY is localized in the prolamellar body fraction in a mostly enzymatically inactive form. Subsequent illumination of dark-grown and/or in far-red light grown seedlings with white light causes the decay of these structures and a topological relocalization of PSY to developing thylakoids which results in its enzymatic activation. This light-dependent mechanism of enzymatic activation of PSY in carotenoid biosynthesis shares common features with the regulation of the NADPH:protochlorophyllide oxidoreductase, the first light-regulated enzyme in chlorophyll biosynthesis. The mechanism of regulation described here may contribute to ensuring a spatially and temporally coordinated increase in both carotenoid and chlorophyll contents. Received: 14 February 2000 / Accepted: 15 March 2000     

PCR identification of durum wheat BAC clones containing genes coding for carotenoid biosynthesis enzymes and their chromosome localization.

Carotenoids are essential components in all plants. Their accumulation in wheat seed determines the endosperm colour, which is an important quality trait in wheat. In this study, we report the isolation of BAC clones containing genes coding for three different enzymes of the carotenoid biosynthesis pathway: phytoene synthase (PSY), phytoene desaturase (PDS), and zeta-carotene desaturase (ZDS). Primers were designed on the basis of wheat ESTs similar to the sequences of these three genes in other species, and used to screen a BAC library from Triticum turgidum var. durum (2n = 28, genomes AABB). Eight, six, and nine 384-well plates containing at least one positive clone were found for PSY, PDS, and ZDS, respectively. BACs selected for each of these genes were then divided in two groups corresponding to the A and B genomes of tetraploid wheat, based on differences in the length of the PCR amplification products, conformation-sensitive gel electrophoresis (CSGE), or cleavage amplification polymorphisms. Positive clones were then assigned to chromosomes using a set of D genome substitution lines in T. turgidum var. durum 'Langdon'. PSY clones were localized on chromosomes 5A and 5B, PDS on chromosomes 4A and 4B, and ZDS on chromosomes 2A and 2B. The strategies used for the PCR screening of large BAC libraries and for the differentiation of BAC clones from different genomes in a polyploid species are discussed.     

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)。     

Budding yeast PDS5 plays an important role in meiosis and is required for sister chromatid cohesion

Budding yeast PDS5 is an essential gene in mitosis and is required for chromosome condensation and sister chromatid cohesion. Here we report that PDS also is required in meiosis. Pds5p localizes on chromosomes at all stages during meiotic cycle, except anaphase I. PDS5 plays an important role at first meiotic prophase. Failure in function of PDS5 causes premature separation of chromosomes. The loading of Pds5p onto chromosome requires the function of REC8, but the association of Rec8p with chromosome is independent of PDS5. Mutant analysis and live cell imaging indicate that PDS5 play a role in meiosis II as well.     

Regulation of carotenoid biosynthesis during fruit maturation in the red-fleshed orange mutant Cara Cara

Cara Cara is a spontaneous bud mutation of Navel orange (Citrus. sinensis L. Osbeck) characterized by developing fruits with a pulp of bright red coloration due to the presence of lycopene. Peel of mutant fruits is however orange and indistinguishable from its parental. To elucidate the basis of lycopene accumulation in Cara Cara, we analyzed carotenoid profile and expression of three isoprenoid and nine carotenoid genes in flavedo and pulp of Cara Cara and Navel fruits throughout development and maturation. The pulp of the mutant accumulated high amounts of lycopene, but also phytoene and phytofluene, from early developmental stages. The peel of Cara Cara also accumulated phytoene and phytofluene. The expression of isoprenoid genes and of carotenoid biosynthetic genes downstream PDS (phytoene desaturase) was higher in the pulp of Cara Cara than in Navel. Not important differences in the expression of these genes were observed between the peel of both oranges. Moreover, the content of the plant hormone ABA (abscisic acid) was lower in the pulp of Cara Cara, but the expression of two genes involved in its biosynthesis was higher. The results suggest that an altered carotenoid composition may conduct to a positive feedback regulatory mechanism of carotenoid biosynthesis in citrus fruits. Increased levels of isoprenoid precursors in the mutant that could be channeled to carotenoid biosynthesis may be related to the red-fleshed phenotype of Cara Cara.     

Metabolic engineering of astaxanthin production in tobacco flowers

Using metabolic engineering, we have modified the carotenoid biosynthesis pathway in tobacco (Nicotiana tabacum) to produce astaxanthin, a red pigment of considerable economic value. To alter the carotenoid pathway in chromoplasts of higher plants, the cDNA of the gene CrtO from the alga Haematococcus pluvialis, encoding beta-carotene ketolase, was transferred to tobacco under the regulation of the tomato Pds (phytoene desaturase) promoter. The transit peptide of PDS from tomato was used to target the CRTO polypeptide to the plastids. Chromoplasts in the nectary tissue of transgenic plants accumulated (3S,3'S) astaxanthin and other ketocarotenoids, changing the color of the nectary from yellow to red. This accomplishment demonstrates that plants can be used as a source of novel carotenoid pigments such as astaxanthin. The procedures described in this work can serve as a platform technology for future genetic manipulations of pigmentation of fruits and flowers of horticultural and floricultural importance.     

Disruption of phytoene desaturase gene results in albino and dwarf phenotypes in Arabidopsis by impairing chlorophyll, carotenoid, and gibberellin biosynthesis

Carotenoids play an important role in many physiological processes in plants and the phytoene desaturase gene （PDS3） encodes one of the important enzymes in the carotenoid biosynthesis pathway. Here we report the identification and analysis of a T-DNA insertion mutant of PDS3 gene. Functional complementation confirmed that both the albino and dwarfphenotypes ofthepds3 mutant resulted from functional disruption of the PDS3 gene. Chloroplast development was arrested at the proplastid stage in thepds3 mutant. Further analysis showed that high level ofphytoene was accumulated in the pds3 mutant. Addition of exogenous GA3 could partially rescue the dwarf phenotype, suggesting that the dwarf phenotype ofthepds3 mutant might be due to GA deficiency. Microarray and RT-PCR analysis showed that disrupting PDS3 gene resulted in gene expression changes involved in at least 20 metabolic pathways, including the inhibition of many genes in carotenoid, chlorophyll, and GA biosynthesis pathways. Our data suggest that the accumulated phytoene in the pds3 mutant might play an important role in certain negative feedbacks to affect gene expression of diverse cellular pathways.