Prenyltransferases from the flavedo of Citrus sinensis

A prenyltransferase activity (EC 2.5.1.1) has been partially purified from the flavedo of Citrus sinensis with 30–40-fold purification and 35–60 % yield. The enzyme catalyses the condensation of IPP with DMAPP or GPP. The products are neryl and geranyl pyrophosphate as well as (2E,6E)- and (2Z,6E)-farnesyl pyrophosphate. The two C₁₅-products are predominant. The E- and Z-synthetase activities are partially dissociated during the purification procedure, as well as by heat or ageing. Preparations devoid of Z-synthetase were obtained. Mg^{2 +} is required for full activity. Mn^{2 +} or Co^{2 +} can replace Mg^{2 +}. The ratio of E/Z-products formed is different for each cation. Mg^{2 +} complexes of allylic substrates or of products protect the enzyme against heat-inactivation and against inactivation by DTNB. The results are interpreted in terms of two or more prenyltransferases stereoselective for the synthesis of E- and Z-products.     

Carotenoid changes in the peel of ripening persimmon (Diospyros kaki) cv Triumph

The changes of the carotenoid pigments in the peel of ripening persimmon (Diospyros kaki) cv Triumph were followed for an entire season. During ripening, the total carotenoid decreased until colour break, then increased gradually and drastically at the last ripening stages. The chloroplast carotenoid pattern of the unripe fruit changed into a chromoplast pattern in which cryptoxanthin was the predominant pigment, reaching a level between 40 and 50% of the total carotenoids. It accumulated continuously at a rate of approximately 10% at each 2 week interval, its percentage being characteristic for each ripening stage. Other major pigments at levels of approximately 10% of the total carotenoids were zeaxanthin, antheraxanthin and violaxanthin. In the fully ripe fruit, ripened both on and off the tree, lycopene which was not present before was found as the second major pigment. This unusual pattern change is discussed.     

Biosynthesis of allylic isoprenoid pyrophosphates by an enzyme preparation from the flavedo of Citrus paradisii

A cell-free system obtained from Citrus paradisii flavedo transformed mevalonic acid into mono- and sesquiterpenoids of E- and Z-conformation. The enzyme system required bivalent metal ions and the presence of sulfhydryl groups. IPP isomerase activity (EC 5.3.3.2) was independent of metal ions and almost insensitive to sulfhydryl group reagents, while prenyltransferase (EC 2.5.1.1) was inactivated by DTNB and required bivalent metals for activity. The nature of the metal ion defined the stereochemistry of the products formed by prenyltransferase. The ratio of E–Z farnesylpyrophosphates was 3:1. This Citrus species could, therefore, be a good starting material for the study of the stereochemistry of the enzymes forming E and Z sesquiterpenoids.     

The stereochemistry of hydroxylation of the carotenoid lutein in Calendula officinalis

Excised, opening inflorescences of Calendula officinalis incorporated (3RS, 5R)- and (3RS, 5S)-[2-¹⁴C,5-³H₁]mevalonates into the carotenoid fraction. The ¹⁴C:³H ratios of lutein isolated from these tissues showed the hydrogen atom at C-3 of the β-ring is derived from the 5-pro-S position of mevalonate, while that at C-3 of the ε-ring is derived from the 5-pro-R position of mevalonate. Oxidation of lutein to monoketolutein showed that both hydrogen atoms at the C-15,15′ central double bond are derived from the 5-pro-R position of mevalonate.     

Hydrolysis of allylic phosphates by enzymes from the flavedo of Citrus sinensis

Acid phosphatase activities have been partially purified from an aqueous extract of an acetone powder from orange flavedo. The use of a gel filtration step with an ionic gradient allowed a dissociation of proteins from pigments, thus facilitating purification and stabilization of the enzymes. The enzymes do not require metals for full activity, and they hydrolysed a wide spectrum of phosphorylated substrates. C₁₀–C₂₀ allylic pyrophosphates and monophosphates were hydrolysed sequentially by these ‘prenylphosphatases’. The final product was the corresponding unrearranged prenyl alcohol. This demonstrated the absence of E-Z isomerization and suggested an OP bond cleavage. Prenylphosphatases exhibited a certain degree of chain length specificity. Although the E or Z conformation of the C-2 double bond was not important, its presence was required for full activity. Excess prenylpyrophosphate inhibited the rate of formation of alcohols, most likely through the inhibition of phosphomonoesterase activity. These prenylphosphatases generated the alcoholic components of essential oils from the corresponding pyrophosphates and removed them from the chain lengthening process.     

Pigment evolution in Lycopersicon esculentum fruits during growth and ripening

During growth and subsequent maturation, the distribution and formation of pigments in the inner pulp and in the outer region of the pericarp of ‘che     

Molecular characterization and expression analysis of ubiquitin-activating enzyme E1 gene in Citrus reticulata

Ubiquitin-activating enzyme E1 (UBE1) catalyzes the first step in the ubiquitination reaction, which targets a protein for degradation via a proteasome pathway. UBE1 plays an important role in metabolic processes. In this study, full-length cDNA and DNA sequences of UBE1 gene, designated CrUBE1, were obtained from ‘Wuzishatangju’ (self-incompatible, SI) and ‘Shatangju’ (self-compatible, SC) mandarins. 5 amino acids and 8 bases were different in cDNA and DNA sequences of CrUBE1 between ‘Wuzishatangju’ and ‘Shatangju’, respectively. Southern blot analysis showed that there existed only one copy of the CrUBE1 gene in genome of ‘Wuzishatangju’ and ‘Shatangju’. The temporal and spatial expression characteristics of the CrUBE1 gene were investigated using semi-quantitative RT-PCR (SqPCR) and quantitative real-time PCR (qPCR). The expression level of the CrUBE1 gene in anthers of ‘Shatangju’ was approximately 10-fold higher than in anthers of ‘Wuzishatangju’. The highest expression level of CrUBE1 was detected in pistils at 7 days after self-pollination of ‘Wuzishatangju’, which was approximately 5-fold higher than at 0 h. To obtain CrUBE1 protein, the full-length cDNA of CrUBE1 genes from ‘Wuzishatangju’ and ‘Shatangju’ were successfully expressed in Pichia pastoris. Pollen germination frequency of ‘Wuzishatangju’ was significantly inhibited with increasing of CrUBE1 protein concentrations from ‘Wuzishatangju’.     

Flower colours and pigments in Disa hybrid (Orchidaceae)

Flower colours and the composition of pigments in the perianths of five cultivars of Disa orchids were analyzed. Carotenoids were major pigment components in the orange-red flowers of ‘Dawn Angel’. We identified two types of pigment composition in the red flowered cultivars: ‘San Francisco’ contained more carotenoids and less anthocyanins, while ‘Marlene’ contained more anthocyanins than carotenoids. The red-purple flowered cultivars, only contained slight amounts of carotenoids, and the red-purple colour was attributed to the relatively high density of a cyanidin-based anthocyanin. The importance of the characterization of pigments in the perianths of orchid has been discussed in both breeding for flower colour improvement and chemotaxonomy.     

The effect of diphenylamine on carotenogenesis in Phycomyces blakesleeanus

The presence of diphenylamine (DPA) during growth of mutant strains of Phycomyces blakesleeanus caused the expected inhibition of the formation of unsaturated carotenes, and the accumulation of phytoene in all cases. Cell extracts from DPA-grown cultures incubated with [2-¹⁴C]mevalonic acid, also exhibited these effects. Inhibition of in vitro carotenogenesis was similarly shown by extracts from normally-grown mycelia, incubated with DPA. Removal of DPA from mycelia or from cell extracts, resulted in the formation of unsaturated carotenes. The ratio of 15-cis-all-trans-phytoenes from mycelia grown ± DPA was only marginally altered, but, in vitro, the presence of DPA caused a significant increase in the formation of the all-trans isomer in the C5 strain. These results indicate that DPA acts by post-translational regulation of enzymic activities.     

Production of somatic hybrids between satsuma mandarin (Citrus unshiu) and navel orange (Citrus sinensis) by protoplast fusion

We have regenerated altotetraploid plants that are interspecific somatic hybrids between Citrus sinensis Osbeck cv. Yoshida navel orange and Citrus unshiu Marc cv. Okitsu satsuma mandarin. Protoplasts isolated from ‘Yoshida’ leaves were chemically fused with call us-derived protoplasts from ‘Okitsu’. After 6 months of culture, 102 plants were obtained. These hybrids were identified by differential leaf morphology, DNA fluorescence intensity, and DNA analysis. Ploidy analysis via the flow cytometry revealed that 15 of the 102 plants were tetraploids, with the rest being diploids that morphologically resembled their mesophyll parent. SRAP analysis confirmed that 9 of the tetraploid plants were allotetraploid somatic hybrids. These will be utilized as a possible pollen parents for improving seedy citrus cultivars, e.g., ponkan, mandarin, lemon and kumquat, in order to produce triploid seedless hybrids.     

The carotenoids of Rhodomicrobium vannielii (rhodospirillaceae) and the effect of diphenylamine on the carotenoid composition

The presence of β-carotene, lycopene, rhodopin, rhodovibrin, anhydrorhodovibrin, spirillox-anthin, ‘monodemethylated spirilloxanthin’ and ‘dihy     

Effect of oleocellosis,desiccation, and fungal infection upon the terpenes of individual oil glands in Citrus latipes

Mono- and sesqui-terpenes of individual oil glands of Citrus latipes fruits were analyzed for their homogeneity. The effect of oleocellosis, desiccation, Penicillium and Phytophthora infection upon the individual terpene components was investigated and expressed in a discriminant analysis and in canonical variables. Each oil gland contained the entire spectrum of terpenes specific for each species, and the biggest difference in affected glands was due to Penicillium infection.     

Biosynthetic relationship of citral-trans and citral-cis in cymbopogon flexuosus (lemongrass)

Use of [¹⁴C,³H]-labelled precursors revealed that leaf blades of Cymbopogon flexuosus converted geraniol (3,7-dimethylocta-trans-2,6-diene-1-ol) into citral-trans with loss of pro-(1S) hydrogen whereas nerol lost the pro-(1R) hydrogen while being converted into citral-cis. Secondly, the citral-trans is converted into citral-cis and vice versa and there is no separate route for the biosynthesis of either of the two aldehyde isomers.     

Fruit carotenoid‐deficient mutants in tomato reveal a function of the plastidial isopentenyl diphosphate isomerase (IDI1) in carotenoid biosynthesis

Isoprenoids consist of a large class of compounds that are present in all living organisms. They are derived from the 5C building blocks isopentenyl diphosphate (IDP) and its isomer dimethylallyl diphosphate (DMADP). In plants, IDP is synthesized in the cytoplasm from mevalonic acid via the MVA pathway, and in plastids from 2‐C‐methyl‐d ‐erythritol‐4‐phosphate through the MEP pathway. The enzyme IDP isomerase (IDI) catalyzes the interconversion between IDP and DMADP. Most plants contain two IDI enzymes, the functions of which are characteristically compartmentalized in the cells. Carotenoids are isoprenoids that play essential roles in photosynthesis and provide colors to flowers and fruits. They are synthesized in the plastids via the MEP pathway. Fruits of Solanum lycopersicum (tomato) accumulate high levels of the red carotene lycopene. We have identified mutations in tomato that reduce overall carotenoid accumulation in fruits. Four alleles of a locus named FRUIT CAROTENOID DEFICIENT 1 (fcd1) were characterized. Map‐based cloning of fcd1 indicated that this gene encodes the plastidial enzyme IDI1. Lack of IDI1 reduced the concentration of carotenoids in fruits, flowers and cotyledons, but not in mature leaves. These results indicate that the plastidial IDI plays an important function in carotenoid biosynthesis, thus highlighting its role in optimizing the ratio between IDP and DMADP as precursors for different downstream isoprenoid pathways.     

Distribution of carotenoids in sub-cellular and sub-plastidic fractions of radish seedlings (Raphanus sativus) grown in the presence of bleaching herbicides

The intracellular and intraplastidic distribution of carotenoids has been investigated in radish seedlings grown in the presence of the herbicides amitrole and SAN 6706. Both herbicides caused bleaching and the plants became deficient in chlorophylls and the usual chloroplast cyclic carotenoids, but accumulated the acyclic carotenoid biosynthetic intermediates 15-cis-phytoene and all-trans-lycopene. In both the untreated and herbicide-treated plants all carotenoids, including phytoene and lycopene, were contained in the plastid. In all cases the normal cyclic carotenoids were located virtually exclusively in the thylakoid or prothylakoid fraction. In amitrole-treated plants, lycopene also was contained only in the thylakoid fraction, whereas phytoene, in these and in SAN 6706-treated plants, was detected in both the thylakoid fraction and an envelope preparation. Possible implications for the biosynthesis of the carotenoids are discussed.     

Dominance hierarchies and factors influencing dominance in the guppy Poecilia reticulata (peters)

Dominance behaviour and factors influencing dominance were investigated in small heterosexual and homosexual groups of guppies (Poecilia reticulata) in the laboratory. Straight line nip-right hierarchies were the most frequent type of dominance relationship. In heterosexual groups separate homosexual hierarchies were formed, and females were the more dominant sex. Dominant males performed significantly more reproductive behaviour than lower rank individuals. Agonistic behaviour appears early in young P. reticulata. Factors involved in determining dominance were size, sex and aggressiveness. Individual or role recognition or both may occur in this species. Advantages of dominance are discussed.     

Hydromineral regulation in the saline water corixid Trichocorixa reticulata (Hemiptera: Corixidae)

The aquatic corixid Trichocorixa reticulata (Guerin-Meneville) inhabits coastal marshes, brackish water ponds and salt ponds of high salinity, suggesting the presence of well developed mechanisms for hydromineral regulation.Groups of corixids acclimated in salinities ranging from fresh water to just above 300% sea water (100‰) were analyzed for total body water content, haemolymph ionic and osmotic levels, and haemolymph free amino acids.Results indicate an excellent ability to maintain haemolymph Na⁺, Cl^?, Mg²⁺ and K⁺ hyperosmotic to the medium at low salinities and hyposmotic at high salinities. Calcium appears to conform closely to changes in external medium, becoming hyposmotic at very high salinities (80‰).Total haemolymph osmotic pressure was well regulated, the freezing point depression varying from 0.75°C in distilled water to 1.15°C in salinities of 100‰. Total body water was maintained at approx. 75% of the total animal wet weight at all salinities tested.Free amino acids were maintained between 40–60 mM in all tests and did not appear to change with salinity.     

Activity,extraction and stability of enzymes involved in polysaccharide biosynthesis in Citrus

The effects of EDTA, 2-mercaptoethanol and bovine serum albumin on the extraction and stability of hexokinase, phosphoglucomutase, UDPglucose pyrophosphorylase and 1,3-β-glucan synthase from Mexican lime bark have been examined. The activity of these enzymes was generally increased and stability was tested in refrigerated and frozen extracts. Bovine serum albumin was the best stabilizing agent for phosphoglucomutase and 1,3-β-glucan synthase, but the former was more stable in refrigerated and the latter in frozen extracts. UDPglucose pyrophosphorylase stability was strongly dependent on the presence of 2-mercaptoethanol. The fact that the activity of 1,3-β-glucan synthase is the lowest of the four enzymes even in the presence of optimal concentrations of its activator strongly suggests that it is the key enzyme in the regulation of the metabolic pathway.