Characterization of Pinalate,a novel Citrus sinensis mutant with a fruit-specific alteration that results in yellow pigmentation and decreased ABA content

The characterization of a novel mutant, named Pinalate, derived from the orange (Citrus sinensis L. Osbeck) Navelate, which produces distinctive yellow fruits instead of the typical bright orange colouration, is reported. The carotenoid content and composition, and ABA content in leaf and flavedo tissue (coloured part of the skin) of fruits at different developmental and maturation stages were analysed. No important differences in leaf carotenoid pattern of both phenotypes were found. However, an unusual accumulation of linear carotenes (phytoene, phytofluene and zeta- carotene) was detected in the flavedo of Pinalate. As fruit maturation progressed, the flavedo of mutant fruit accumulated high amounts of these carotenes and the proportion of cyclic and oxygenated carotenoids was substantially lower than in the parental line. Full-coloured fruit of Pinalate contained about 44% phytoene, 21% phytofluene, 25% zeta-carotene, and 10% of xanthophylls, whereas, in Navelate, 98% of total carotenoids were xanthophylls and apocarotenoids. The ABA content in the flavedo of Pinalate mature fruit was 3-6 times lower than in the corresponding tissue of Navelate, while no differences were found in leaves. Other maturation processes were not affected in Pinalate fruit. Taken together, the results indicate that Pinalate is a fruit-specific alteration defective in zeta-carotene desaturase or in zeta-carotene desaturase-associated factors. Possible mechanisms responsible for the Pinalate phenotype are discussed. Because of the abnormal fruit-specific carotenoid complement and ABA deficiency, Pinalate may constitute an excellent system for the study of carotenogenesis in Citrus and the involvement of ABA in fruit maturation and stress responses.     

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.     

Identification of the carotenoid isomerase provides insight into carotenoid biosynthesis, prolamellar body formation, and photomorphogenesis

Carotenoids are essential photoprotective and antioxidant pigments synthesized by all photosynthetic organisms. Most carotenoid biosynthetic enzymes were thought to have evolved independently in bacteria and plants. For example, in bacteria, a single enzyme (CrtI) catalyzes the four desaturations leading from the colorless compound phytoene to the red compound lycopene, whereas plants require two desaturases (phytoene and zeta-carotene desaturases) that are unrelated to the bacterial enzyme. We have demonstrated that carotenoid desaturation in plants requires a third distinct enzyme activity, the carotenoid isomerase (CRTISO), which, unlike phytoene and zeta-carotene desaturases, apparently arose from a progenitor bacterial desaturase. The Arabidopsis CRTISO locus was identified by the partial inhibition of lutein synthesis in light-grown tissue and the accumulation of poly-cis-carotene precursors in dark-grown tissue of crtISO mutants. After positional cloning, enzymatic analysis of CRTISO expressed in Escherichia coli confirmed that the enzyme catalyzes the isomerization of poly-cis-carotenoids to all-trans-carotenoids. Etioplasts of dark-grown crtISO mutants accumulate acyclic poly-cis-carotenoids in place of cyclic all-trans-xanthophylls and also lack prolamellar bodies (PLBs), the lattice of tubular membranes that defines an etioplast. This demonstrates a requirement for carotenoid biosynthesis to form the PLB. The absence of PLBs in crtISO mutants demonstrates a function for this unique structure and carotenoids in facilitating chloroplast development during the first critical days of seedling germination and photomorphogenesis.     

Why Is Golden Rice Golden (Yellow) Instead of Red?

The endosperm of Golden Rice (Oryza sativa) is yellow due to the accumulation of beta-carotene (provitamin A) and xanthophylls. The product of the two carotenoid biosynthesis transgenes used in Golden Rice, phytoene synthase (PSY) and the bacterial carotene desaturase (CRTI), is lycopene, which has a red color. The absence of lycopene in Golden Rice shows that the pathway proceeds beyond the transgenic end point and thus that the endogenous pathway must also be acting. By using TaqMan real-time PCR, we show in wild-type rice endosperm the mRNA expression of the relevant carotenoid biosynthetic enzymes encoding phytoene desaturase, zeta-carotene desaturase, carotene cis-trans-isomerase, beta-lycopene cyclase, and beta-carotene hydroxylase; only PSY mRNA was virtually absent. We show that the transgenic phenotype is not due to up-regulation of expression of the endogenous rice pathway in response to the transgenes, as was suggested to be the case in tomato (Lycopersicon esculentum) fruit, where CRTI expression resulted in a similar carotenoid phenomenon. This means that beta-carotene and xanthophyll formation in Golden Rice relies on the activity of constitutively expressed intrinsic rice genes (carotene cis-trans-isomerase, alpha/beta-lycopene cyclase, beta-carotene hydroxylase). PSY needs to be supplemented and the need for the CrtI transgene in Golden Rice is presumably due to insufficient activity of the phytoene desaturase and/or zeta-carotene desaturase enzyme in endosperm. The effect of CRTI expression was also investigated in leaves of transgenic rice and Arabidopsis (Arabidopsis thaliana). Here, again, the mRNA levels of intrinsic carotenogenic enzymes remained unaffected; nevertheless, the carotenoid pattern changed, showing a decrease in lutein, while the beta-carotene-derived xanthophylls increased. This shift correlated with CRTI-expression and is most likely governed at the enzyme level by lycopene-cis-trans-isomerism. Possible implications are discussed.     

Bleaching activity of 4-phenyl-3-(substituted benzylthio)-4H-1,2,4-triazoles

A variety of 4-aryl- and 4-alkyl-3-(substituted benzylthio)-4H-1,2,4-triazoles were prepared and evaluated for their bleaching activity by the lettuce seedling test. Among the series of tested compounds, 4-(3-fluorophenyl)-3-(4-trifluoromethylbenzylthio)-4H-1,2,4-triazole (39) exhibited the highest bleaching activity, causing complete bleaching symptoms at 10 microM. In the dark condition, compound 39 inhibited the formation of such carotenoids as beta-carotene, violaxanthin, neoxanthin and lutein, resulting in the formation of zeta-carotene, phytoene, phytofluene and beta-zeacarotene, which were not detected in the untreated control. Treatment by compound 39 at 50 microM resulted in the amount of accumulated zeta-carotene being seven-fold higher than that of phytoene, phytofluene and beta-zeacarotene. These results suggest that compound 39 might have interfered with desaturation, especially zeta-carotene desaturation, during carotenoid biosynthesis.     

A plastid terminal oxidase associated with carotenoid desaturation during chromoplast differentiation

The Arabidopsis IMMUTANS gene encodes a plastid homolog of the mitochondrial alternative oxidase, which is associated with phytoene desaturation. Upon expression in Escherichia coli, this protein confers a detectable cyanide-resistant electron transport to isolated membranes. In this assay this activity is sensitive to n-propyl-gallate, an inhibitor of the alternative oxidase. This protein appears to be a plastid terminal oxidase (PTOX) that is functionally equivalent to a quinol:oxygen oxidoreductase. This protein was immunodetected in achlorophyllous pepper (Capsicum annuum) chromoplast membranes, and a corresponding cDNA was cloned from pepper and tomato (Lycopersicum esculentum) fruits. Genomic analysis suggests the presence of a single gene in these organisms, the expression of which parallels phytoene desaturase and ζ-carotene desaturase gene expression during fruit ripening. Furthermore, this PTOX gene is impaired in the tomato ghost mutant, which accumulates phytoene in leaves and fruits. These data show that PTOX also participates in carotenoid desaturation in chromoplasts in addition to its role during early chloroplast development.     

Biosynthesis of acyclic and cyclic carotenoids in higher plants and the control by phytochrome

Radish plants ( Raphanus sativus L. cv. Saxa treib) were grown in the presence of three different herbicides interfering with the biosynthesis of cyclic carotenoids. The herbicides caused an accumulation of acyclic biosynthetic intermediates. Plants were then irradiated using four different light programs in order to gain more insight into the first steps of carotenoid biosynthesis and their control by light and phytochrome. Plants grown in the dark in the presence of SAN 6706 or aminotriazole accumulated the acyclic intermediate phytoene, and those treated with J 852, the intermediates phytoene, phytofluene and zeta-carotene. In herbicide-treated plants short time irradiation with red light enhanced the formation of phytoene, phytofluene, zeta-carotene or lycopene, consistent with an effect of phytochrome on the early steps of carotenoid biosynthesis. Biosynthesis of cyclic carotenoids was also enhanced by red light in the untreated controls. In amitrole-treated plants formation of β-carotene, but not that of xanthophylls was stimulated by red light. In many cases neither the red light-induced biosynthesis of cyclic carotenoids nor the formation of acyclic intermediates could be prevented by a subsequent irradiation with far-red light. Similar enhancement as with red light was also obtained after treatment with far-red light only. Presented data may be taken as evidence that the biosynthesis and dehydrogenation of phytoene and the cyclization of lycopene are activated by a low threshold of active phytochrome. This may be further supported by the observation that far-red light itself stimulated carotenoid biosynthesis.     

Bansformation of tobacco with a mutated cyanobacterial phytoene desaturase gene confers resistance to bleaching herbicides

Carotenoids are constituents of the photosynthetic apparatus and essential for plant survival because of their involvement in protection of chlorophylls against photooxidation. Certain classes of herbicides are interfering with carotenoid biosynthesis leading to pigment destruction and a bleached plant phenotype. One important target site for bleaching herbicides is the enzyme phytoene desaturase catalysing the desaturation of phytoene in zeta-carotene. This enzymatic reaction can be inhibited by norflurazon or fluridone. We have transformed tobacco with a mutated cyanobacterial phytoene desaturase gene (pds) derived from the Synechococcus PCC 7942 mutant NFZ4. Characterization of the resulting transformants revealed an up to 58 fold higher norflurazon resistance in comparison to wild type controls. The tolerance for fluridone was also increased 3 fold in the transgenics. Furthermore, the transformed tobacco maintained a higher level of D1 protein of photosystem II indicating a lower susceptibility to photooxidative damage in the presence of norflurazon. In contrast, the genetic manipulation did not confer herbicide resistance against zeta-carotene desaturase inhibitors.     

Transformation of the green alga Haematococcus pluvialis with a phytoene desaturase for accelerated astaxanthin biosynthesis

Astaxanthin is a high-value carotenoid which is used as a pigmentation source in fish aquaculture. Additionally, a beneficial role of astaxanthin as a food supplement for humans has been suggested. The unicellular alga Haematococcus pluvialis is a suitable biological source for astaxanthin production. In the context of the strong biotechnological relevance of H. pluvialis, we developed a genetic transformation protocol for metabolic engineering of this green alga. First, the gene coding for the carotenoid biosynthesis enzyme phytoene desaturase was isolated from H. pluvialis and modified by site-directed mutagenesis, changing the leucine codon at position 504 to an arginine codon. In an in vitro assay, the modified phytoene desaturase was still active in conversion of phytoene to zeta-carotene and exhibited 43-fold-higher resistance to the bleaching herbicide norflurazon. Upon biolistic transformation using the modified phytoene desaturase gene as a reporter and selection with norflurazon, integration into the nuclear genome of H. pluvialis and phytoene desaturase gene and protein expression were demonstrated by Southern, Northern, and Western blotting, respectively, in 11 transformants. Some of the transformants had a higher carotenoid content in the green state, which correlated with increased nonphotochemical quenching. This measurement of chlorophyll fluorescence can be used as a screening procedure for stable transformants. Stress induction of astaxanthin biosynthesis by high light showed that there was accelerated accumulation of astaxanthin in one of the transformants compared to the accumulation in the wild type. Our results strongly indicate that the modified phytoene desaturase gene is a useful tool for genetic engineering of carotenoid biosynthesis in H. pluvialis.     

Identification of a cDNA for the plastid-located geranylgeranyl pyrophosphate synthase from Capsicum annuum: correlative increase in enzyme activity and transcript level during fruit ripening

Geranylgeranyl pyrophosphate synthase is a key enzyme in plant terpenoid biosynthesis. Using specific antibodies, a cDNA encoding geranylgeranyl pyrophosphate synthase has been isolated from bell pepper (Capsicum annuum) ripening fruit. The cloned cDNA codes for a high molecular weight precursor of 369 amino acids which contains a transit peptide of approximately 60 amino acids. In-situ immunolocalization experiments have demonstrated that geranylgeranyl pyrophosphate synthase is located exclusively in the plastids. Expression of the cloned cDNA in E. coli has unambiguously demonstrated that the encoded polypeptide catalyzes the synthesis of geranylgeranyl pyrophosphate by the addition of isopentenyl pyrophosphate to an allylic pyrophosphate. Peptide sequence comparisons revealed significant similarity between the sequences of the C. annuum geranylgeranyl pyrophosphate synthase and those deduced from carotenoid biosynthesis (crtE) genes from photosynthetic and non-photosynthetic bacteria. In addition, four highly conserved regions, which are found in various prenyltransferases, were identified. Furthermore, evidence is provided suggesting that conserved and exposed carboxylates are directly involved in the catalytic mechanism. Finally, the expression of the geranylgeranyl pyrophosphate synthase gene is demonstrated to be strongly induced during the chloroplast to chromoplast transition which occurs in ripening fruits, and is correlated with an increase in enzyme activity.     

Interallelic complementation provides genetic evidence for the multimeric organization of the Phycomyces blakesleeanus phytoene dehydrogenase.

The Phycomyces blakesleeanus wild-type is yellow, because it accumulates beta-carotene as the main carotenoid. A new carotenoid mutant of this fungus (A486) was isolated, after treatment with ethyl methane sulfonate (EMS), showing a whitish coloration. It accumulates large amounts of phytoene, small quantities of phytofluene, zeta-carotene and neurosporene, in decreasing amounts, and traces of beta-carotene. This phenotype indicates that it carries a leaky mutation affecting the enzyme phytoene dehydrogenase (EC 1.3.-.-), which is specified by the gene carB. Biochemical analysis of heterokaryons showed that mutant A486 complements two previously characterized carB mutants, C5 (carB10) and S442 (carB401). Sequence analysis of the carB gene genomic copy from these three strains revealed that they are all altered in the gene carB, giving information about the nature of the mutation in each carB mutant allele. The interallelic complementation provides evidence for the multimeric organization of the P. blakesleeanus phytoene dehydrogenase.     

Functional properties of diapophytoene and related desaturases of C(30) and C(40) carotenoid biosynthetic pathways

The desaturation reactions of C(30) carotenoids from diapophytoene to diaponeurosporene was investigated in vitro and by complementation in Escherichia coli. The expressed diapophytoene desaturase from Staphylococcus aureus inserts three double bonds in an FAD-dependent reaction. The enzyme is inhibited by diphenylamine. In the complementation experiment diapophytoene desaturase was able to convert C(40) phytoene to some extend but exhibited a high affinity to zeta-carotene. Comparison to the reaction of a phytoene desaturase from Rhodobacter capsulatus catalyzing a parallel three-step desaturation sequence with the corresponding C(40) carotenes revealed that this desaturase can also convert C(30) diapophytoene. Other homologous bacterial C(40) carotene desaturases could also utilize C(30) substrates, including one type of zeta-carotene desaturase which converted diaponeurosporene to diapolycopene. Further complementation experiments including the diapophytoene synthase gene from S. aureus revealed that the C(30) carotenogenic pathway is determined by this initial enzyme which is highly homologous to C(40) phytoene synthases.     

Cooperation of two carotene desaturases in the production of lycopene in Myxococcus xanthus

In Myxococcus xanthus, all known carotenogenic genes are grouped together in the gene cluster carB-carA, except for one, crtIb (previously named carC). We show here that the first three genes of the carB operon, crtE, crtIa, and crtB, encode a geranygeranyl synthase, a phytoene desaturase, and a phytoene synthase, respectively. We demonstrate also that CrtIa possesses cis-to-trans isomerase activity, and is able to dehydrogenate phytoene, producing phytofluene and zeta-carotene. Unlike the majority of CrtI-type phytoene desaturases, CrtIa is unable to perform the four dehydrogenation events involved in converting phytoene to lycopene. CrtIb, on the other hand, is incapable of dehydrogenating phytoene and lacks cis-to-trans isomerase activity. However, the presence of both CrtIa and CrtIb allows the completion of the four desaturation steps that convert phytoene to lycopene. Therefore, we report a unique mechanism where two distinct CrtI-type desaturases cooperate to carry out the four desaturation steps required for lycopene formation. In addition, we show that there is a difference in substrate recognition between the two desaturases; CrtIa dehydrogenates carotenes in the cis conformation, whereas CrtIb dehydrogenates carotenes in the trans conformation.     

Phytoene synthase from Narcissus pseudonarcissus: functional expression, galactolipid requirement, topological distribution in chromoplasts and induction during flowering

A cDNA coding for the carotenoid biosynthetic enzyme phytoene synthase was cloned from a Narcissus pseudonarcissus flower cDNA library, and the corresponding protein was overexpressed in insect cells using the baculovirus lipofection system. The full-length overexpressed enzyme exhibited very reduced catalytic activity compared with an overexpressed N-truncated form, with its transit sequence removed by site-directed mutagenesis. The shortened form readily bound quantitatively to lipid bilayers. Although it was active with liposomes prepared from plastid lipids, with phospholipid liposomes it was not, even though association took place. In this latter case, free galactose was capable of substituting for galactolipids, resulting in enzymatic activity. It is concluded that galactolipids are involved in catalytic activity, but do not serve as a membrane anchor. Antibodies raised against the recombinant enzyme made it possible to distinguish between a membrane-bound and a soluble, protein-complexed inactive form of phytoene synthase, present in the chromoplast stroma. These findings and data on phytoene synthase mRNA and protein expression presented here are discussed in terms of a possible regulatory role in color formation during chromoplast (flower) development.     

Chromoplast differentiation in bell pepper (Capsicum annuum) fruits

We report here a detailed analysis of the proteome adjustments that accompany chromoplast differentiation from chloroplasts during bell pepper (Capsicum annuum) fruit ripening. While the two photosystems are disassembled and their constituents degraded, the cytochrome b₆f complex, the ATPase complex, and Calvin cycle enzymes are maintained at high levels up to fully mature chromoplasts. This is also true for ferredoxin (Fd) and Fd-dependent NADP reductase, suggesting that ferredoxin retains a central role in the chromoplasts’ redox metabolism. There is a significant increase in the amount of enzymes of the typical metabolism of heterotrophic plastids, such as the oxidative pentose phosphate pathway (OPPP) and amino acid and fatty acid biosynthesis. Enzymes of chlorophyll catabolism and carotenoid biosynthesis increase in abundance, supporting the pigment reorganization that goes together with chromoplast differentiation. The majority of plastid encoded proteins decline but constituents of the plastid ribosome and AccD increase in abundance. Furthermore, the amount of plastid terminal oxidase (PTOX) remains unchanged despite a significant increase in phytoene desaturase (PDS) levels, suggesting that the electrons from phytoene desaturation are consumed by another oxidase. This may be a particularity of non-climacteric fruits such as bell pepper that lack a respiratory burst at the onset of fruit ripening.