Dually biofortified cisgenic tomatoes with increased flavonoids and branched-chain amino acids content.

Higher dietary intakes of flavonoids may have a beneficial role in cardiovascular disease prevention. Additionally, supplementation of branched-chain amino acids (BCAAs) in vegan diets can reduce risks associated to their deficiency, particularly in older adults, which can cause loss of skeletal muscle strength and mass. Most plant-derived foods contain only small amounts of BCAAs, and those plants with high levels of flavonoids are not eaten broadly. Here we describe the generation of metabolically engineered cisgenic tomatoes enriched in both flavonoids and BCAAs. In this approach, coding and regulatory DNA elements, all derived from the tomato genome, were combined to obtain a herbicide-resistant version of an acetolactate synthase (mSlALS) gene expressed broadly and a MYB12-like transcription factor (SlMYB12) expressed in a fruit-specific manner. The mSlALS played a dual role, as a selectable marker as well as being key enzyme in BCAA enrichment. The resulting cisgenic tomatoes were highly enriched in Leucine (21-fold compared to wild-type levels), Valine (ninefold) and Isoleucine (threefold) and concomitantly biofortified in several antioxidant flavonoids including kaempferol (64-fold) and quercetin (45-fold). Comprehensive metabolomic and transcriptomic analysis of the biofortified cisgenic tomatoes revealed marked differences to wild type and could serve to evaluate the safety of these biofortified fruits for human consumption.

Aglianico Grape Seed Semi-Polar Extract Exerts Anticancer Effects by Modulating MDM2 Expression and Metabolic Pathways.

Grapevine (Vitis vinifera L.) seeds are rich in polyphenols including proanthocyanidins, molecules with a variety of biological effects including anticancer action. We have previously reported that the grape seed semi-polar extract of Aglianico cultivar (AGS) was able to induce apoptosis and decrease cancer properties in different mesothelioma cell lines. Concomitantly, this extract resulted in enriched oligomeric proanthocyanidins which might be involved in determining the anticancer activity. Through transcriptomic and metabolomic analyses, we investigated in detail the anticancer pathway induced by AGS. Transcriptomics analysis and functional annotation allowed the identification of the relevant causative genes involved in the apoptotic induction following AGS treatment. Subsequent biological validation strengthened the hypothesis that MDM2 could be the molecular target of AGS and that it could act in both a p53-dependent and independent manner. Finally, AGS significantly inhibited tumor progression in a xenograft mouse model of mesothelioma, confirming also in vivo that MDM2 could act as molecular player responsible for the AGS antitumor effect. Our findings indicated that AGS, exerting a pro-apoptotic effect by hindering MDM2 pathway, could represent a novel source of anticancer molecules.

Fortification and bioaccessibility of saffron apocarotenoids in potato tubers.

Carotenoids are C40 isoprenoids with well-established roles in photosynthesis, pollination, photoprotection, and hormone biosynthesis. The enzymatic or ROS-induced cleavage of carotenoids generates a group of compounds named apocarotenoids, with an increasing interest by virtue of their metabolic, physiological, and ecological activities. Both classes are used industrially in a variety of fields as colorants, supplements, and bio-actives. Crocins and picrocrocin, two saffron apocarotenoids, are examples of high-value pigments utilized in the food, feed, and pharmaceutical industries. In this study, a unique construct was achieved, namely O6, which contains CsCCD2L, UGT74AD1, and UGT709G1 genes responsible for the biosynthesis of saffron apocarotenoids driven by a patatin promoter for the generation of potato tubers producing crocins and picrocrocin. Different tuber potatoes accumulated crocins and picrocrocin ranging from 19.41-360 to 105-800 µg/g DW, respectively, with crocetin, crocin 1 [(crocetin-(ß-D-glucosyl)-ester)] and crocin 2 [(crocetin)-(ß-D-glucosyl)-(ß-D-glucosyl)-ester)] being the main compounds detected. The pattern of carotenoids and apocarotenoids were distinct between wild type and transgenic tubers and were related to changes in the expression of the pathway genes, especially from PSY2, CCD1, and CCD4. In addition, the engineered tubers showed higher antioxidant capacity, up to almost 4-fold more than the wild type, which is a promising sign for the potential health advantages of these lines. In order to better investigate these aspects, different cooking methods were applied, and each process displayed a significant impact on the retention of apocarotenoids. More in detail, the in vitro bioaccessibility of these metabolites was found to be higher in boiled potatoes (97.23%) compared to raw, baked, and fried ones (80.97, 78.96, and 76.18%, respectively). Overall, this work shows that potatoes can be engineered to accumulate saffron apocarotenoids that, when consumed, can potentially offer better health benefits. Moreover, the high bioaccessibility of these compounds revealed that potato is an excellent way to deliver crocins and picrocrocin, while also helping to improve its nutritional value.

Liver Steatosis and Steatohepatitis Alter Bile Acid Receptors in Brain and Induce Neuroinflammation: A Contribution of Circulating Bile Acids and Blood-Brain Barrier.

A tight relationship between gut-liver diseases and brain functions has recently emerged. Bile acid (BA) receptors, bacterial-derived molecules and the blood-brain barrier (BBB) play key roles in this association. This study was aimed to evaluate how non-alcoholic fatty liver disease (NAFLD) and non-alcoholic steatohepatitis (NASH) impact the BA receptors Farnesoid X receptor (FXR) and Takeda G-protein coupled receptor 5 (TGR5) expression in the brain and to correlate these effects with circulating BAs composition, BBB integrity and neuroinflammation. A mouse model of NAFLD was set up by a high-fat and sugar diet, and NASH was induced with the supplementation of dextran-sulfate-sodium (DSS) in drinking water. FXR, TGR5 and ionized calcium-binding adaptor molecule 1 (Iba-1) expression in the brain was detected by immunohistochemistry, while Zonula occludens (ZO)-1, Occludin and Plasmalemmal Vesicle Associated Protein-1 (PV-1) were analyzed by immunofluorescence. Biochemical analyses investigated serum BA composition, lipopolysaccharide-binding protein (LBP) and S100ß protein (S100ß) levels. Results showed a down-regulation of FXR in NASH and an up-regulation of TGR5 and Iba-1 in the cortex and hippocampus in both treated groups as compared to the control group. The BA composition was altered in the serum of both treated groups, and LBP and S100ß were significantly augmented in NASH. ZO-1 and Occludin were attenuated in the brain capillary endothelial cells of both treated groups versus the control group. We demonstrated that NAFLD and NASH provoke different grades of brain dysfunction, which are characterized by the altered expression of BA receptors, FXR and TGR5, and activation of microglia. These effects are somewhat promoted by a modification of circulating BAs composition and by an increase in LBP that concur to damage BBB, thus favoring neuroinflammation.

Comparative chemical analysis of six ancient italian sweet cherry (Prunus avium L.) varieties showing antiangiogenic activity.

In this study, cherry fruits and petioles from six ancient Italian Prunus avium L. varieties (Ferrovia, Capellina, Morellina, Ciambellana, Napoletana, and Bianca), were compared by chemical and bioinformatic analyses and evaluated for their antiangiogenic activity. The highest levels of total phenols and flavonoids were found in Napoletana petioles, and Morellina and Capellina fruits. HPLC-PDA-MS analyses showed similar phenolic profiles for all fruit extracts, with cyanidin-3-O-rutinoside, flavonols glycosides, and quinic acid derivatives as major components. Flavonoid glycosides were found in all petiole extracts, while proanthocyanidins B type were predominant in Capellina, Napoletana and Bianca. Accordingly to their higher polyphenolic content, petiole extracts exhibited stronger radical scavenging activity compared to the fruits. The best antiangiogenic response was exhibited by Morellina, Ferrovia, and Ciambellana petiole extracts, and by Ferrovia, Morellina, and Capellina fruit extracts; by bioinformatic studies rutin and cyanidin 3-O-rutinoside were recognised as the best candidate bioactive compounds. In conclusion, sweet cherry varietes were confirmed as valuable sources of phenols, showing also potential angiomodulator properties.

ABCC Transporters Mediate the Vacuolar Accumulation of Crocins in Saffron Stigmas.

Compartmentation is a key strategy enacted by plants for the storage of specialized metabolites. The saffron spice owes its red color to crocins, a complex mixture of apocarotenoid glycosides that accumulate in intracellular vacuoles and reach up to 10% of the spice dry weight. We developed a general approach, based on coexpression analysis, heterologous expression in yeast (Saccharomyces cerevisiae), and in vitro transportomic assays using yeast microsomes and total plant metabolite extracts, for the identification of putative vacuolar metabolite transporters, and we used it to identify Crocus sativus transporters mediating vacuolar crocin accumulation in stigmas. Three transporters, belonging to both the multidrug and toxic compound extrusion and ATP binding cassette C (ABCC) families, were coexpressed with crocins and/or with the gene encoding the first dedicated enzyme in the crocin biosynthetic pathway, CsCCD2. Two of these, belonging to the ABCC family, were able to mediate transport of several crocins when expressed in yeast microsomes. CsABCC4a was selectively expressed in C. sativus stigmas, was predominantly tonoplast localized, transported crocins in vitro in a stereospecific and cooperative way, and was able to enhance crocin accumulation when expressed in Nicotiana benthamiana leaves.plantcell;31/11/2789/FX1F1fx1.

UGT709G1: a novel uridine diphosphate glycosyltransferase involved in the biosynthesis of picrocrocin, the precursor of safranal in saffron (Crocus sativus).

Saffron, a spice derived from the dried red stigmas of Crocus sativus, is one of the oldest natural food additives. The flowers have long red stigmas, which store significant quantities of the glycosylated apocarotenoids crocins and picrocrocin. The apocarotenoid biosynthetic pathway in saffron starts with the oxidative cleavage of zeaxanthin, from which crocins and picrocrocin are derived. In the processed stigmas, picrocrocin is converted to safranal, giving saffron its typical aroma. By a targeted search for differentially expressed uridine diphosphate glycosyltransferases (UGTs) in Crocus transcriptomes, a novel apocarotenoid glucosyltransferase (UGT709G1) from saffron was identified. Biochemical analyses revealed that UGT709G1 showed a high catalytic efficiency toward 2,6,6-trimethyl-4-hydroxy-1-carboxaldehyde-1-cyclohexene (HTCC), making it suited for the biosynthesis of picrocrocin, the precursor of safranal. The role of UGT709G1 in picrocrocin/safranal biosynthesis was supported by the absence or presence of gene expression in a screening for HTCC and picrocrocin production in different Crocus species and by a combined transient expression assay with CsCCD2L in Nicotiana benthamiana leaves. The identification of UGT709G1 completes one of the most highly valued specialized metabolic biosynthetic pathways in plants and provides novel perspectives on the industrial production of picrocrocin to be used as a flavor additive or as a pharmacological constituent.

Potential of golden potatoes to improve vitamin A and vitamin E status in developing countries.

Potato (Solanum tuberosum L.) is the third most widely consumed plant food by humans. Its tubers are rich in starch and vitamin C, but have low or null levels of essential nutrients such as provitamin A and vitamin E. Transformation of potato with a bacterial mini-pathway for ß-carotene in a tuber-specific manner results in a "golden" potato (GP) tuber phenotype resulting from accumulation of provitamin A carotenoids (α- and ß-carotene) and xanthophylls. Here, we investigated the bioaccessibility of carotenoids and vitamin E as α-tocopherol (αTC) in boiled wild type and golden tubers using in vitro digestion. Golden tubers contained up to 91 µg provitamin A carotenes (PAC)/g D, increased levels of xanthophylls, phytoene and phytofluene, as well as up to 78 µg vitamin E/g DW. Cubes from wild type and GP tubers were boiled and subjected to simulated digestion to estimate bioaccessibility of carotenoids and αTC. Retention in boiled GPs exceeded 80% for ß-carotene (ßC), α-carotene (αC), lutein, phytoene ± and αTC, but less than 50% for phytofluene. The efficiency of partitioning of total ßC, αC, E-lutein, phytoene, phytofluene and αTC in the mixed micelle fraction during small intestinal digestion was influenced by genotype, tuber content and hydrophobicity. Apical uptake of the compounds that partitioned in mixed micelles by monolayers of human intestinal Caco-2 cells during incubation for 4h was 14-20% for provitamin A and xanthophylls, 43-45% for phytoene, 23-27% for phytofluene, and 53% for αTC. These results suggest that a 150 g serving of boiled golden potatoes has the potential to contribute 42% and 23% of the daily requirement of retinol activity equivalents (RAE), as well as 34 and 17% of the daily vitamin E requirement for children and women of reproductive age, respectively.

Molecular and biochemical characterization of a potato collection with contrasting tuber carotenoid content.

After wheat and rice, potato is the third most important staple food worldwide. A collection of ten tetraploid (Solanum tuberosum) and diploid (S. phureja and S. chacoense) genotypes with contrasting carotenoid content was subjected to molecular characterization with respect to candidate carotenoid loci and metabolic profiling using LC-HRMS. Irrespective of ploidy and taxonomy, tubers of these genotypes fell into three groups: yellow-fleshed, characterized by high levels of epoxy-xanthophylls and xanthophyll esters and by the presence of at least one copy of a dominant allele of the ß-Carotene Hydroxylase 2 (CHY2) gene; white-fleshed, characterized by low carotenoid levels and by the presence of recessive chy2 alleles; and orange-fleshed, characterized by high levels of zeaxanthin but low levels of xanthophyll esters, and homozygosity for a Zeaxanthin Epoxidase (ZEP) recessive allele. Novel CHY2 and ZEP alleles were identified in the collection. Multivariate analysis identified several groups of co-regulated non-polar compounds, and resulted in the grouping of the genotypes according to flesh color, suggesting that extensive cross-talk exists between the carotenoid pathway and other metabolite pathways in tubers. Postharvest traits like tuber dormancy and weight loss during storage showed little correlation with tuber carotenoid content, with the exception of zeaxanthin and its esters. Other tuber metabolites, such as glucose, monogalactosyldiacyglycerol (a glycolipid), or suberin precursors, showed instead significant correlations with both traits.

Tissue-Specific Accumulation of Sulfur Compounds and Saponins in Different Parts of Garlic Cloves from Purple and White Ecotypes.

This study set out to determine the distribution of sulfur compounds and saponin metabolites in different parts of garlic cloves. Three fractions from purple and white garlic ecotypes were obtained: the tunic (SS), internal (IS) and external (ES) parts of the clove. Liquid Chromatography coupled to High Resolution Mass spectrometry (LC-HRMS), together with bioinformatics including Principal Component Analysis (PCA), Hierarchical Clustering (HCL) and correlation network analyses were carried out. Results showed that the distribution of these metabolites in the different parts of garlic bulbs was different for the purple and the white ecotypes, with the main difference being a slightly higher number of sulfur compounds in purple garlic. The SS fraction in purple garlic had a higher content of sulfur metabolites, while the ES in white garlic was more enriched by these compounds. The correlation network indicated that diallyl disulfide was the most relevant metabolite with regards to sulfur compound metabolism in garlic. The total number of saponins was almost 40-fold higher in purple garlic than in the white variety, with ES having the highest content. Interestingly, five saponins including desgalactotigonin-rhamnose, proto-desgalactotigonin, proto-desgalactotigonin-rhamnose, voghieroside D1, sativoside B1-rhamnose and sativoside R1 were exclusive to the purple variety. Data obtained from saponin analyses revealed a very different network between white and purple garlic, thus suggesting a very robust and tight coregulation of saponin metabolism in garlic. Findings in this study point to the possibility of using tunics from purple garlic in the food and medical industries, since it contains many functional compounds which can be exploited as ingredients.

Comparative metabolite and genome analysis of tuber-bearing potato species.

The cultivated potato Solanum tuberosum is unrivalled among crop plants for its wild relatives, which potentially represent an important source of genetic diversity to improve the nutritional value of potato varieties and understand metabolism regulation. The main aim of this research was to profile human health-related metabolites in a number of clones from 13 Solanum species. Results from HPLC-DAD and LC-ESI-MS analyses highlighted a high interspecific variability in the level of metabolites analysed. Ascorbic acid was confirmed to be the most abundant antioxidant in potato and chlorogenic acid the primary polyphenol. Generally, metabolite-based hierarchical clustering (HCL) and correlation networks did not group clones of identical species in the same cluster. This might be due to various factors, including the outcrossing nature of potato species, gene expression level and metabolic profiling techniques. Access to the genome sequence of S. tuberosum and S. commersonii allowed comparison of the genes involved in ascorbic acid, aromatic amino acid, phenylpropanoid and glycoalkaloid biosynthesis and helped interpret their respective pathways.

Transcriptome and metabolome of synthetic Solanum autotetraploids reveal key genomic stress events following polyploidization.

Polyploids are generally classified as autopolyploids, derived from a single species, and allopolyploids, arising from interspecific hybridization. The former represent ideal materials with which to study the consequences of genome doubling and ascertain whether there are molecular and functional rules operating following polyploidization events. To investigate whether the effects of autopolyploidization are common to different species, or if species-specific or stochastic events are prevalent, we performed a comprehensive transcriptomic and metabolomic characterization of diploids and autotetraploids of Solanum commersonii and Solanum bulbocastanum. Autopolyploidization remodelled the transcriptome and the metabolome of both species. In S. commersonii, differentially expressed genes (DEGs) were highly enriched in pericentromeric regions. Most changes were stochastic, suggesting a strong genotypic response. However, a set of robustly regulated transcripts and metabolites was also detected, including purine bases and nucleosides, which are likely to underlie a common response to polyploidization. We hypothesize that autopolyploidization results in nucleotide pool imbalance, which in turn triggers a genomic shock responsible for the stochastic events observed. The more extensive genomic stress and the higher number of stochastic events observed in S. commersonii with respect to S. bulbocastanum could be the result of the higher nucleoside depletion observed in this species.

Transcriptional-metabolic networks in beta-carotene-enriched potato tubers: the long and winding road to the Golden phenotype.

Vitamin A deficiency is a public health problem in a large number of countries. Biofortification of major staple crops (wheat [Triticum aestivum], rice [Oryza sativa], maize [Zea mays], and potato [Solanum tuberosum]) with ß-carotene has the potential to alleviate this nutritional problem. Previously, we engineered transgenic "Golden" potato tubers overexpressing three bacterial genes for ß-carotene synthesis (CrtB, CrtI, and CrtY, encoding phytoene synthase, phytoene desaturase, and lycopene ß-cyclase, respectively) and accumulating the highest amount of ß-carotene in the four aforementioned crops. Here, we report the systematic quantitation of carotenoid metabolites and transcripts in 24 lines carrying six different transgene combinations under the control of the 35S and Patatin (Pat) promoters. Low levels of B-I expression are sufficient for interfering with leaf carotenogenesis, but not for ß-carotene accumulation in tubers and calli, which requires high expression levels of all three genes under the control of the Pat promoter. Tubers expressing the B-I transgenes show large perturbations in the transcription of endogenous carotenoid genes, with only minor changes in carotenoid content, while the opposite phenotype (low levels of transcriptional perturbation and high carotenoid levels) is observed in Golden (Y-B-I) tubers. We used hierarchical clustering and pairwise correlation analysis, together with a new method for network correlation analysis, developed for this purpose, to assess the perturbations in transcript and metabolite levels in transgenic leaves and tubers. Through a "guilt-by-profiling" approach, we identified several endogenous genes for carotenoid biosynthesis likely to play a key regulatory role in Golden tubers, which are candidates for manipulations aimed at the further optimization of tuber carotenoid content.

Biosynthesis and engineering of carotenoids and apocarotenoids in plants: state of the art and future prospects.

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. An update on plant carotenoid engineering is also provided for non-solanaceous crops and members of the Solanaceae family, by means of different strategies and making use of plant and bacterial genes.

Metabolic engineering of potato carotenoid content through tuber-specific overexpression of a bacterial mini-pathway.

BACKGROUND: Since the creation of "Golden Rice", biofortification of plant-derived foods is a promising strategy for the alleviation of nutritional deficiencies. Potato is the most important staple food for mankind after the cereals rice, wheat and maize, and is extremely poor in provitamin A carotenoids. METHODOLOGY: We transformed potato with a mini-pathway of bacterial origin, driving the synthesis of beta-carotene (Provitamin A) from geranylgeranyl diphosphate. Three genes, encoding phytoene synthase (CrtB), phytoene desaturase (CrtI) and lycopene beta-cyclase (CrtY) from Erwinia, under tuber-specific or constitutive promoter control, were used. 86 independent transgenic lines, containing six different promoter/gene combinations, were produced and analyzed. Extensive regulatory effects on the expression of endogenous genes for carotenoid biosynthesis are observed in transgenic lines. Constitutive expression of the CrtY and/or CrtI genes interferes with the establishment of transgenosis and with the accumulation of leaf carotenoids. Expression of all three genes, under tuber-specific promoter control, results in tubers with a deep yellow ("golden") phenotype without any adverse leaf phenotypes. In these tubers, carotenoids increase approx. 20-fold, to 114 mcg/g dry weight and beta-carotene 3600-fold, to 47 mcg/g dry weight. CONCLUSIONS: This is the highest carotenoid and beta-carotene content reported for biofortified potato as well as for any of the four major staple foods (the next best event being "Golden Rice 2", with 31 mcg/g dry weight beta-carotene). Assuming a beta-carotene to retinol conversion of 6ratio1, this is sufficient to provide 50% of the Recommended Daily Allowance of Vitamin A with 250 gms (fresh weight) of "golden" potatoes.

Silencing of beta-carotene hydroxylase increases total carotenoid and beta-carotene levels in potato tubers.

BACKGROUND: Beta-carotene is the main dietary precursor of vitamin A. Potato tubers contain low levels of carotenoids, composed mainly of the xanthophylls lutein (in the beta-epsilon branch) and violaxanthin (in the beta-beta branch). None of these carotenoids have provitamin A activity. We have previously shown that tuber-specific silencing of the first step in the epsilon-beta branch, LCY-e, redirects metabolic flux towards beta-beta carotenoids, increases total carotenoids up to 2.5-fold and beta-carotene up to 14-fold. RESULTS: In this work, we silenced the non-heme beta-carotene hydroxylases CHY1 and CHY2 in the tuber. Real Time RT-PCR measurements confirmed the tuber-specific silencing of both genes . CHY silenced tubers showed more dramatic changes in carotenoid content than LCY-e silenced tubers, with beta-carotene increasing up to 38-fold and total carotenoids up to 4.5-fold. These changes were accompanied by a decrease in the immediate product of beta-carotene hydroxylation, zeaxanthin, but not of the downstream xanthophylls, viola- and neoxanthin. Changes in endogenous gene expression were extensive and partially overlapping with those of LCY-e silenced tubers: CrtISO, LCY-b and ZEP were induced in both cases, indicating that they may respond to the balance between individual carotenoid species. CONCLUSION: Together with epsilon-cyclization of lycopene, beta-carotene hydroxylation is another regulatory step in potato tuber carotenogenesis. The data are consistent with a prevalent role of CHY2, which is highly expressed in tubers, in the control of this step. Combination of different engineering strategies holds good promise for the manipulation of tuber carotenoid content.

Metabolic engineering of potato tuber carotenoids through tuber-specific silencing of lycopene epsilon cyclase.

BACKGROUND: Potato is a major staple food, and modification of its provitamin content is a possible means for alleviating nutritional deficiencies. beta-carotene is the main dietary precursor of vitamin A. Potato tubers contain low levels of carotenoids, composed mainly of the xanthophylls lutein, antheraxanthin, violaxanthin, and of xanthophyll esters. None of these carotenoids have provitamin A activity. RESULTS: We silenced the first dedicated step in the beta-epsilon- branch of carotenoid biosynthesis, lycopene epsilon cyclase (LCY-e), by introducing, via Agrobacterium-mediated transformation, an antisense fragment of this gene under the control of the patatin promoter. Real Time measurements confirmed the tuber-specific silencing of Lcy-e. Antisense tubers showed significant increases in beta-beta-carotenoid levels, with beta-carotene showing the maximum increase (up to 14-fold). Total carotenoids increased up to 2.5-fold. These changes were not accompanied by a decrease in lutein, suggesting that LCY-e is not rate-limiting for lutein accumulation. Tuber-specific changes in expression of several genes in the pathway were observed. CONCLUSION: The data suggest that epsilon-cyclization of lycopene is a key regulatory step in potato tuber carotenogenesis. Upon tuber-specific silencing of the corresponding gene, beta-beta-carotenoid and total carotenoid levels are increased, and expression of several other genes in the pathway is modified.