Carlo Rosati

Metabolic engineering of xanthophyll content in tomato fruits

Ripe tomato fruits accumulate significant amounts of the linear carotene lycopene, but only trace amounts of xanthophylls (oxygenated carotenoids). We overexpressed the lycopene β‐cyclase (b‐Lcy) and β‐carotene hydroxylase (b‐Chy) genes under the control of the fruit‐specific Pds promoter. Transgene and protein expression was followed through semi‐quantitative reverse transcription‐PCR, Western blotting, and enzyme assays. Fruits of the transformants showed a significant increase of β‐carotene, β‐cryptoxanthin and zeaxanthin. The carotenoid composition of leaves remained unaltered. The transgenes and the phenotype are inherited in a dominant Mendelian fashion. This is the first example of successful metabolic engineering of xanthophyll content in tomato fruits.

Developmental, genetic and environmental factors affect the expression of flavonoid genes, enzymes and metabolites in strawberry fruits*

The influence of internal (genetic and developmental) and external (environmental) factors on levels of flavonoid gene transcripts, enzyme activity and metabolites was studied in fruit of six cultivated strawberry (Fragaria x ananassa Duch.) genotypes grown at two Italian locations. Gene expression and enzyme activity showed development- and genotype-associated patterns, revealing gene coordination. Analysis clarified the regulation mechanism of the hydroxylation status of the B-ring of the major flavonoid pools and pointed out examples of genotype-specific post-transcriptional regulation mechanisms and key steps of pathway regulation in strawberry fruits. Metabolite profiles were strongly affected by development and genotype. Flavan-3-ols, their proanthocyanidin (PA) derivatives and anthocyanins were the most abundant metabolites. Flavonol levels and PA-associated traits (epicatechin/catechin ratio and mean degree of polymerization) showed significant environmental effects. Multivariate and correlation analyses determined the relationships among genes, enzymes and metabolites. The combined molecular and biochemical information elucidated more in depth the role of genetic and environmental factors on flavonoid metabolism during strawberry fruit development, highlighting the major impact of developmental processes, and revealing genotype-dependent differences and environmental effects on PA-related traits.

Carotenoid isomerase: a tale of light and isomers

Carotenoids are terpenoid pigments containing systems of conjugated double bonds, each of which can exist in a cis or trans configuration. The existence of a carotenoid isomerase (CrtISO) mediating cis-to-trans isomerization has been suspected ever since the description of the tomato tangerine mutant in the early 1940s, whose fruits accumulate a poly-cis-isomer of lycopene. Recently, CrtISO has been cloned in Synechocystis and in plants, and has been shown to be related to bacterial carotene desaturases.

Erratum: Engineering of flower color in forsythia by expression of two independently-transformed dihydroflavonol 4-reductase and anthocyanidin synthase genes of flavonoid pathway

Flower color was modified in forsythia (Forsythia x intermedia cv ‘Spring Glory’) by inducing anthocyanin synthesis in petals through sequential Agrobacterium-mediated transformation with dihydroflavonol 4-reductase from Antirrhinum majus (AmDFR) and anthocyanidin synthase from Matthiola incana (MiANS) genes. This is the second report of flower color modification of an ornamental shrub after rose, and the first time an ANS gene is used for this purpose. Double transformants (AmDFR+MiANS) displayed a novel bronze-orange petal color, caused by the de novo accumulation of cyanidin-derived anthocyanins over the carotenoid yellow background of wild type (wt), and intense pigmentation of vegetative organs. Transformation with single genes (either AmDFR or MiANS) produced no change in flower color, showing a multistep control of late anthocyanin pathway in petals of forsythia. Analysis of relevant late flavonoid pathway genes – an endogenous flavonoid glycosyltransferase (FiFGT) and transformed DFR and ANS genes – showed appropriate expression in flower organs. Functional characterization of FiFGT expressed in E. coli revealed its ability to metabolize both flavonols and anthocyanidin substrates, a prerequisite for effective anthocyanin accumulation in petals of plants transformed with constructs leading to anthocyanidin synthesis. Biochemical analyses of flavonoid compounds in petals and leaves showed that, besides anthocyanin induction in petals of double transformants, the accumulation pattern of flavan-3-ols was quantitatively and qualitatively modified in petals and leaves of transformants, in agreement with the most recent model proposed for flavan-3-ol synthesis. On the other hand, phenylpropanoid, flavone and flavonol pools were not quantitatively affected, indicating a tight regulation of early flavonoid pathway.

Molecular cloning and expression analysis of dihydroflavonol 4-reductase gene in flower organs of Forsythia x intermedia.

The expression, during flower development, of the gene encoding the anthocyanin pathway key enzyme dihydroflavonol 4-reductase (DFR) was investigated in floral organs of Forsythia × intermedia cv. ‘Spring Glory’. Full-length DFR and partial chalcone synthase (CHS) cDNAs, the gene of interest and a flavonoid pathway control gene respectively, were obtained from petal RNA by reverse transcription PCR. Whereas for CHS northern blot analysis enabled the study of its expression pattern, competitive PCR assays were necessary to quantify DFR mRNA levels in wild-type plants and in petals of 2 transgenic clones containing a CaMV 35S promoter-driven DFR gene of Antirrhinum majus. Results indicated a peak of CHS and DFR transcript levels in petals at the very early stages of anthesis, and different expression patterns in anthers and sepals. In comparison to wild-type plants, transformants showed a more intense anthocyanin pigmentation of some vegetative organs, and a dramatic increase in DFR transcript concentration and enzymatic activity in petals. However, petals of transformed plants did not accumulate any anthocyanins. These results indicate that other genes and/or regulatory factors should be considered responsible for the lack of anthocyanin production in Forsythia petals.

Identifying a Carotenoid Cleavage Dioxygenase (ccd4) Gene Controlling Yellow/White Fruit Flesh Color of Peach

Peach flesh color is a monogenic trait with the white phenotype being dominant over the yellow; its expression has been reported to be determined by a carotenoid degradative enzyme. In the present study, a carotenoid cleavage dioxygenase (ccd4) gene was analyzed to test whether it can be responsible for the flesh color determinism. The analysis was conducted on chimeric mutants with white and yellow sectors of the fruit mesocarp; it was then extended to a pool of cultivars and a segregating F1 population. A ccd4 functional allele is consistently associated with the ancestral white flesh color; on the other hand, the yellow phenotype originated from at least three independent mutations disrupting ccd4 function, thus preventing carotenoid degradation. In addition, retro-mutations recovering ccd4 function and re-establishing the ancestral white flesh color were detected. Our results show that ccd4 is the gene controlling flesh color in peach; its expression results in the degradation of carotenoids in white-fleshed genotypes, while the yellow color arises as a consequence of its inactivation.

Biosynthesis and Engineering of Carotenoids and Apocarotenoids in Plants: State of the Art and Future Prospects

Abstract Carotenoids and their apocarotenoid derivatives are isoprenoid molecules important for the primary and secondary metabolisms of plants and other living organisms, displaying also key health-related roles in humans and animals. Progress in the knowledge of the carotenoid pathway at the genetic, biochemical and molecular level, supported by successful genetic engineering examples for an increasing number of important plant crops have paved the way for precise molecular breeding of carotenoids. In this review, following a description of the general carotenoid pathway, select examples of plant species able to produce specialty carotenoids and apocarotenoids are illustrated.

Molecular characterization of the anthocyanidin synthase gene in Forsythia intermedia reveals organ-specific expression during flower development

The study of the anthocyanidin synthase (ANS) gene, one of the late structural genes in the anthocyanin pathway, was undertaken in Forsythiaintermedia cv. ‘Spring Glory’. Previous molecular and biochemical studies had demonstrated expression and activity of genes and enzymes upstream of ans. The ans gene was cloned and shown to be present in two copies in the Forsythia genome. Expression analyses carried out on flower organs showed that ans was expressed exclusively in anthocyanincontaining sepals and not in anthocyaninless anthers and petals. ans expression in sepals showed induction of transcription at early flower developmental stages. Inspection of the ans promoter region as far as 790 bp upstream of the start codon revealed several potential DNA‐protein binding motifs. The results from this paper, combined with previous data, show that the lack of ans expression should be the major cause of the absence of anthocyanins in Forsythia petals, thus providing directions for genetic engineering of flower color in this ornamental species.

Volatile emissions of scented Alstroemeria genotypes are dominated by terpenes, and a myrcene synthase gene is highly expressed in scented Alstroemeria flowers

Native to South America, Alstroemeria flowers are known for their colourful tepals, and Alstroemeria hybrids are an important cut flower. However, in common with many commercial cut flowers, virtually all the commercial Alstroemeria hybrids are not scented. The cultivar ‘Sweet Laura’ is one of very few scented commercial Alstroemeria hybrids. Characterization of the volatile emission profile of these cut flowers revealed three major terpene compounds: (E)-caryophyllene, humulene (also known as α-caryophyllene), an ocimene-like compound, and several minor peaks, one of which was identified as myrcene. The profile is completely different from that of the parental scented species A. caryophyllaea. Volatile emission peaked at anthesis in both scented genotypes, coincident in cv. ‘Sweet Laura’ with the maximal expression of a putative terpene synthase gene AlstroTPS. This gene was preferentially expressed in floral tissues of both cv. ‘Sweet Laura’ and A. caryophyllaea. Characterization of the AlstroTPS gene structure from cv. ‘Sweet Laura’ placed it as a member of the class III terpene synthases, and the predicted 567 amino acid sequence placed it into the subfamily TPS-b. The conserved sequences R28(R)X8W and D321DXXD are the putative Mg2+-binding sites, and in vitro assay of AlstroTPS expressed in Escherichia coli revealed that the encoded enzyme possesses myrcene synthase activity, consistent with a role for AlstroTPS in scent production in Alstroemeria cv. ‘Sweet Laura’ flowers.

Flavonoid metabolism in Forsythia flowers

Abstract The flavonoid pathway metabolism was studied in petals and sepals of Forsythia X intermedia cv. ‘Spring Glory’. The activities of the flavonoid enzymes chalcone synthase (CHS), flavanone 3-hydroxylase (FHT) and flavonol synthase (FLS) were measured. The dihydroflavonol 4-reductase (DFR) role was also studied by comparing flavonoid accumulation in transgenic plants for a heterologous DFR gene and wild-type F. X intermedia cv. ‘Spring Glory’, already investigated for DFR gene expression and activity. HPLC analyses complemented enzymatic investigations, showing that: (i) rutin (quercetin 3-rutinoside) is the major flavonol accumulated in petals and sepals (ca. 90% of the flavonol pool) and; (ii) quercetin and cyanidin derivatives are the exclusive flavonols and anthocyanins in sepals, respectively. The overall data demonstrated that the flavonoid pathway in F. X intermedia flower organs leads to the major accumulation of 3′,4′-dihydroxylated compounds, and that 3′-hydroxylation of the B-ring occurs mainly at flavonoid intermediate stage(s). Comparative HPLC analyses of F. X intermedia cv. ‘Spring Glory’ and three other genotypes ( F. giraldiana , F. X intermedia ‘Korfor’ Goldzauber® and F. ovata ‘Robusta’) confirmed the major production of flavonols by Forsythia flavonoid metabolism and suggested a method for screening Forsythia genotypes based on anthocyanin accumulation pattern in sepals.