Carotenoid biosynthesis genes LcLCYB, LcLCYE, and LcBCH from wolfberry confer increased carotenoid content and improved salt tolerance in tobacco.

Carotenoids play essential roles in plant growth and development and provide plants with a tolerance to a series of abiotic stresses. In this study, the function and biological significance of lycopene ß-cyclase, lycopene ε-cyclase, and ß-carotene hydroxylase, which are responsible for the modification of the tetraterpene skeleton procedure, were isolated from Lycium chinense and analyzed. The overexpression of lycopene ß-cyclase, lycopene ε-cyclase, and ß-carotene hydroxylase promoted the accumulation of total carotenoids and photosynthesis enhancement, reactive oxygen species scavenging activity, and proline content of tobacco seedlings after exposure to the salt stress. Furthermore, the expression of the carotenoid biosynthesis genes and stress-related genes (ascorbate peroxidase, catalase, peroxidase, superoxide dismutase, and pyrroline-5-carboxylate reductase) were detected and showed increased gene expression level, which were strongly associated with the carotenoid content and reactive oxygen species scavenging activity. After exposure to salt stress, the endogenous abscisic acid content was significantly increased and much higher than those in control plants. This research contributes to the development of new breeding aimed at obtaining stronger salt tolerance plants with increased total carotenoids and vitamin A content.

Functional Characterization of Lycopene ß- and ε-Cyclases from a Lutein-Enriched Green Microalga Chlorella sorokiniana FZU60.

Lutein is a high-value carotenoid with many human health benefits. Lycopene ß- and ε-cyclases (LCYB and LCYE, respectively) catalyze the cyclization of lycopene into distinct downstream branches, one of which is the lutein biosynthesis pathway, via α-carotene. Hence, LCYB and LCYE are key enzymes in lutein biosynthesis. In this study, the coding genes of two lycopene cyclases (CsLCYB and CsLCYE) of a lutein-enriched marine green microalga, Chlorella sorokiniana FZU60, were isolated and identified. A sequence analysis and computational modeling of CsLCYB and CsLCYE were performed using bioinformatics to identify the key structural domains. Further, a phylogenetic analysis revealed that CsLCYB and CsLCYE were homogeneous to the proteins of other green microalgae. Subcellular localization tests in Nicotiana benthamiana showed that CsLCYB and CsLCYE localized in chloroplasts. A pigment complementation assay in Escherichia coli revealed that CsLCYB could efficiently ß-cyclize both ends of lycopene to produce ß-carotene. On the other hand, CsLCYE possessed a strong ε-monocyclase activity for the production of δ-carotene and a weak ε-bicyclic activity for the production of ε-carotene. In addition, CsLCYE was able to catalyze lycopene into ß-monocyclic γ-carotene and ultimately produced α-carotene with a ß-ring and an ε-ring via γ-carotene or δ-carotene. Moreover, the co-expression of CsLCYB and CsLCYE in E. coli revealed that α-carotene was a major product, which might lead to the production of a high level of lutein in C. sorokiniana FZU60. The findings provide a theoretical foundation for performing metabolic engineering to improve lutein biosynthesis and accumulation in C. sorokiniana FZU60.

Regulatory ligand binding in plant chalcone isomerase-like (CHIL) proteins.

Chalcone isomerase-like (CHIL) protein is a noncatalytic protein that enhances flavonoid content in green plants by serving as a metabolite binder and a rectifier of chalcone synthase (CHS). Rectification of CHS catalysis occurs through direct protein-protein interactions between CHIL and CHS, which alter CHS kinetics and product profiles, favoring naringenin chalcone (NC) production. These discoveries raise questions about how CHIL proteins interact structurally with metabolites and how CHIL-ligand interactions affect interactions with CHS. Using differential scanning fluorimetry on a CHIL protein from Vitis vinifera (VvCHIL), we report that positive thermostability effects are induced by the binding of NC, and negative thermostability effects are induced by the binding of naringenin. NC further causes positive changes to CHIL-CHS binding, whereas naringenin causes negative changes to VvCHIL-CHS binding. These results suggest that CHILs may act as sensors for ligand-mediated pathway feedback by influencing CHS function. The protein X-ray crystal structure of VvCHIL compared with the protein X-ray crystal structure of a CHIL from Physcomitrella patens reveals key amino acid differences at a ligand-binding site of VvCHIL that can be substituted to nullify the destabilizing effect caused by naringenin. Together, these results support a role for CHIL proteins as metabolite sensors that modulate the committed step of the flavonoid pathway.

Aim18p and Aim46p are chalcone isomerase domain-containing mitochondrial hemoproteins in Saccharomyces cerevisiae.

Chalcone isomerases (CHIs) have well-established roles in the biosynthesis of plant flavonoid metabolites. Saccharomyces cerevisiae possesses two predicted CHI-like proteins, Aim18p (encoded by YHR198C) and Aim46p (YHR199C), but it lacks other enzymes of the flavonoid pathway, suggesting that Aim18p and Aim46p employ the CHI fold for distinct purposes. Here, we demonstrate using proteinase K protection assays, sodium carbonate extractions, and crystallography that Aim18p and Aim46p reside on the mitochondrial inner membrane and adopt CHI folds, but they lack select active site residues and possess an extra fungal-specific loop. Consistent with these differences, Aim18p and Aim46p lack CHI activity and also the fatty acid-binding capabilities of other CHI-like proteins, but instead bind heme. We further show that diverse fungal homologs also bind heme and that Aim18p and Aim46p possess structural homology to a bacterial hemoprotein. Collectively, our work reveals a distinct function and cellular localization for two CHI-like proteins, introduces a new variation of a hemoprotein fold, and suggests that ancestral CHI-like proteins were hemoproteins.

Genome-Wide Classification and Evolutionary Analysis Reveal Diverged Patterns of Chalcone Isomerase in Plants.

Flavonoids as a class of important secondary metabolites are widely present in land plants, and chalcone isomerase (CHI) is the key rate-limiting enzyme that participates in catalyzing the stereospecific isomerization of chalcones to yield their corresponding flavanones. However, the phylogenetic dynamics and functional divergence of CHI family genes during the evolutionary path of green plants remains poorly understood. Here, a total of 122 CHI genes were identified by performing a genome-wide survey of 15 representative green plants from the most ancestral basal plant chlorophyte algae to higher angiosperm plants. Phylogenetic, orthologous groups (OG) classification, and genome structure analysis showed that the CHI family genes have evolved into four distinct types (types I-IV) containing eight OGs after gene duplication, and further studies indicated type III CHIs consist of three subfamilies (FAP1, FAP2, and FAP3). The phylogeny showed FAP3 CHIs as an ancestral out-group positioned on the outer layers of the main branch, followed by type IV CHIs, which are placed in an evolutionary intermediate between FAP3 CHIs and bona fide CHIs (including type I and type II). The results imply a potential intrinsic evolutionary connection between CHIs existing in the green plants. The amino acid substitutions occurring in several residues have potentially affected the functional divergence between CHI proteins. This is supported by the analysis of transcriptional divergence and cis-acting element analysis. Evolutionary dynamics analyses revealed that the differences in the total number of CHI family genes in each plant are primarily attributed to the lineage-specific expansion by natural selective forces. The current studies provide a deeper understanding of the phylogenetic relationships and functional diversification of CHI family genes in green plants, which will guide further investigation on molecular characteristics and biological functions of CHIs.

Removal of lycopene substrate inhibition enables high carotenoid productivity in Yarrowia lipolytica.

Substrate inhibition of enzymes can be a major obstacle to the production of valuable chemicals in engineered microorganisms. Here, we show substrate inhibition of lycopene cyclase as the main limitation in carotenoid biosynthesis in Yarrowia lipolytica. To overcome this bottleneck, we exploit two independent approaches. Structure-guided protein engineering yields a variant, Y27R, characterized by complete loss of substrate inhibition without reduction of enzymatic activity. Alternatively, establishing a geranylgeranyl pyrophosphate synthase-mediated flux flow restrictor also prevents the onset of substrate inhibition by diverting metabolic flux away from the inhibitory metabolite while maintaining sufficient flux towards product formation. Both approaches result in high levels of near-exclusive ß-carotene production. Ultimately, we construct strains capable of producing 39.5 g/L ß-carotene at a productivity of 0.165 g/L/h in bioreactor fermentations (a 1441-fold improvement over the initial strain). Our findings provide effective approaches for removing substrate inhibition in engineering pathways for efficient synthesis of natural products.

Analyses of key gene networks controlling carotenoid metabolism in Xiangfen 1 banana.

BACKGROUND: Banana fruits are rich in various high-value metabolites and play a key role in the human diet. Of these components, carotenoids have attracted considerable attention due to their physiological role and human health care functions. However, the accumulation patterns of carotenoids and genome-wide analysis of gene expression during banana fruit development have not been comprehensively evaluated. RESULTS: In the present study, an integrative analysis of metabolites and transcriptome profiles in banana fruit with three different development stages was performed. A total of 11 carotenoid compounds were identified, and most of these compounds showed markedly higher abundances in mature green and/or mature fruit than in young fruit. Results were linked to the high expression of carotenoid synthesis and regulatory genes in the middle and late stages of fruit development. Co-expression network analysis revealed that 79 differentially expressed transcription factor genes may be responsible for the regulation of LCYB (lycopene ß-cyclase), a key enzyme catalyzing the biosynthesis of α- and ß-carotene. CONCLUSIONS: Collectively, the study provided new insights into the understanding of dynamic changes in carotenoid content and gene expression level during banana fruit development.

Mutant combinations of lycopene ɛ-cyclase and ß-carotene hydroxylase 2 homoeologs increased ß-carotene accumulation in endosperm of tetraploid wheat (Triticum turgidum L.) grains.

Grains of tetraploid wheat (Triticum turgidum L.) mainly accumulate the non-provitamin A carotenoid lutein-with low natural variation in provitamin A ß-carotene in wheat accessions necessitating alternative strategies for provitamin A biofortification. Lycopene ɛ-cyclase (LCYe) and ß-carotene hydroxylase (HYD) function in diverting carbons from ß-carotene to lutein biosynthesis and catalyzing the turnover of ß-carotene to xanthophylls, respectively. However, the contribution of LCYe and HYD gene homoeologs to carotenoid metabolism and how they can be manipulated to increase ß-carotene in tetraploid wheat endosperm (flour) is currently unclear. We isolated loss-of-function Targeting Induced Local Lesions in Genomes (TILLING) mutants of LCYe and HYD2 homoeologs and generated higher order mutant combinations of lcye-A, lcye-B, hyd-A2, and hyd-B2. Hyd-A2 hyd-B2, lcye-A hyd-A2 hyd-B2, lcye-B hyd-A2 hyd-B2, and lcye-A lcye-B hyd-A2 hyd-B2 achieved significantly increased ß-carotene in endosperm, with lcye-A hyd-A2 hyd-B2 exhibiting comparable photosynthetic performance and light response to control plants. Comparative analysis of carotenoid profiles suggests that eliminating HYD2 homoeologs is sufficient to prevent ß-carotene conversion to xanthophylls in the endosperm without compromising xanthophyll production in leaves, and that ß-carotene and its derived xanthophylls are likely subject to differential catalysis mechanisms in vegetative tissues and grains. Carotenoid and gene expression analyses also suggest that the very low LCYe-B expression in endosperm is adequate for lutein production in the absence of LCYe-A. These results demonstrate the success of provitamin A biofortification using TILLING mutants while also providing a roadmap for guiding a gene editing-based approach in hexaploid wheat.

Identification and characterization of unique 5-hydroxyisoflavonoid biosynthetic key enzyme genes in Lupinus albus.

KEY MESSAGE: 5-Hydroxyisoflavonoids, no 5-deoxyisoflavonoids, in Lupinus species, are due to lack of CHRs and Type II CHIs, and the key enzymes of isoflavonoid biosynthetic pathway in white lupin were identified. White lupin (Lupinus albus) is used as food ingredients owing to rich protein, low starch, and rich bioactive compounds such as isoflavonoids. The isoflavonoids biosynthetic pathway in white lupin still remains unclear. In this study, only 5-hydroxyisoflavonoids, but no 5-deoxyisoflavonoids, were detected in white lupin and other Lupinus species. No 5-deoxyisoflavonoids in Lupinus species are due to lack of CHRs and Type II CHIs. We further found that the CHI gene cluster containing both Type I and Type II CHIs possibly arose after the divergence of Lupinus with other legume clade. LaCHI1 and LaCHI2 identified from white lupin metabolized naringenin chalcone to naringenin in yeast and tobacco (Nicotiana benthamiana), and were bona fide Type I CHIs. We further identified two isoflavone synthases (LaIFS1 and LaIFS2), catalyzing flavanone naringenin into isoflavone genistein and also catalyzing liquiritigenin into daidzein in yeast and tobacco. In addition, LaG6DT1 and LaG6DT2 prenylated genistein at the C-6 position into wighteone. Two glucosyltransferases LaUGT1 and LaUGT2 metabolized genistein and wighteone into its 7-O-glucosides. Taken together, our study not only revealed that exclusive 5-hydroxyisoflavonoids do exist in Lupinus species, but also identified key enzymes in the isoflavonoid biosynthetic pathway in white lupin.

Identification of Chalcone Isomerase Family Genes and Roles of CnCHI4 in Flavonoid Metabolism in Camellia nitidissima.

Camellia nitidissima is a woody plant with high ornamental value, and its golden-yellow flowers are rich in a variety of bioactive substances, especially flavonoids, that are beneficial to human health. Chalcone isomerases (CHIs) are key enzymes in the flavonoid biosynthesis pathway; however, there is a scarcity of information regarding the CHI family genes of C. nitidissima. In this study, seven CHI genes of C. nitidissima were identified and divided into three subfamilies by phylogenetic analysis. The results of multiple sequence alignment revealed that, unlike CnCHI1/5/6/7, CnCHI2/3/4 are bona fide CHIs that contain all the active site and critical catalytic residues. Analysis of the expression patterns of CnCHIs and the total flavonoid content of the flowers at different developmental stages revealed that CnCHI4 might play an essential role in the flavonoid biosynthesis pathway of C. nitidissima. CnCHI4 overexpression significantly increased flavonoid production in Nicotiana tabacum and C. nitidissima. The results of the dual-luciferase reporter assay and yeast one-hybrid system revealed that CnMYB7 was the key transcription factor that governed the transcription of CnCHI4. The study provides a comprehensive understanding of the CHI family genes of C. nitidissima and performed a preliminary analysis of their functions and regulatory mechanisms.

Elusive partners: a review of the auxiliary proteins guiding metabolic flux in flavonoid biosynthesis.

Flavonoids are specialized metabolites widely distributed across the plant kingdom. They are involved in the growth and survival of plants, conferring the ability to filter ultra-violet rays, conduct symbiotic partnerships, and respond to stress. While many branches of flavonoid biosynthesis have been resolved, recent discoveries suggest missing auxiliary components. These overlooked elements can guide metabolic flux, enhance production, mediate stereoselectivity, transport intermediates, and exert regulatory functions. This review describes several families of auxiliary proteins from across the plant kingdom, including examples from specialized metabolism. In flavonoid biosynthesis, we discuss the example of chalcone isomerase-like (CHIL) proteins and their non-catalytic role. CHILs mediate the cyclization of tetraketides, forming the chalcone scaffold by interacting with chalcone synthase (CHS). Loss of CHIL activity leads to derailment of the CHS-catalyzed reaction and a loss of pigmentation in fruits and flowers. Similarly, members of the pathogenesis-related 10 (PR10) protein family have been found to differentially bind flavonoid intermediates, guiding the composition of anthocyanins. This role comes within a larger body of PR10 involvement in specialized metabolism, from outright catalysis (e.g., (S)-norcoclaurine synthesis) to controlling stereochemistry (e.g., enhancing cis-trans cyclization in catnip). Both CHILs and PR10s hail from larger families of ligand-binding proteins with a spectrum of activity, complicating the characterization of their enigmatic roles. Strategies for the discovery of auxiliary proteins are discussed, as well as mechanistic models for their function. Targeting such unanticipated components will be crucial in manipulating plants or engineering microbial systems for natural product synthesis.

Comparative transcriptome analysis of unripe and ripe banana (cv. Nendran) unraveling genes involved in ripening and other related processes.

Banana is one of the most important fruit crops consumed globally owing to its high nutritional value. Previously, we demonstrated that the ripe pulp of the banana cultivar (cv.) Nendran (AAB) contained a high amount of pro-vitamin A carotenoids. However, the molecular factors involved in the ripening process in Nendran fruit are unexplored. Hence, we commenced a transcriptome study by using the Illumina HiSeq 2500 at two stages i.e. unripe and ripe fruit-pulp of Nendran. Overall, 3474 up and 4727 down-regulated genes were obtained. A large number of identified transcripts were related to genes involved in ripening, cell wall degradation and aroma formation. Gene ontology analysis highlighted differentially expressed genes that play a key role in various pathways. These pathways were mainly linked to cellular, molecular and biological processes. The present transcriptome study also reveals a crucial role of up-regulated carotenoid biosynthesis pathway genes namely, lycopene beta cyclase and geranylgeranyl pyrophosphate synthase at the ripening stage. Genes related to the ripening and other processes like aroma and flavor were highly expressed in the ripe pulp. Expression of numerous transcription factor family genes was also identified. This study lays a path towards understanding the ripening, carotenoid accumulation and other related processes in banana.

Marker-assisted pyramiding of lycopene-ε-cyclase, ß-carotene hydroxylase1 and opaque2 genes for development of biofortified maize hybrids.

Malnutrition affects growth and development in humans and causes socio-economic losses. Normal maize is deficient in essential amino acids, lysine and tryptophan; and vitamin-A. Crop biofortification is a sustainable and economical approach to alleviate micronutrient malnutrition. We combined favorable alleles of crtRB1 and lcyE genes into opaque2 (o2)-based four inbreds viz. QLM11, QLM12, QLM13, and QLM14 using marker-assisted backcross breeding. These are parents of quality protein maize versions of two elite hybrids viz. Buland and PMH1, grown in India. Gene-based SSRs for o2 and InDel markers for crtRB1 and lcyE were successfully employed for foreground selection in BC1F1, BC2F1, and BC2F2 generations. The recurrent parent genome recovery ranged from 88.9 to 96.0% among introgressed progenies. Kernels of pyramided lines possessed a high concentration of proA (7.14-9.63 ppm), compared to 1.05 to 1.41 ppm in the recurrent parents, while lysine and tryptophan ranged from 0.28-0.44% and 0.07-0.09%, respectively. The reconstituted hybrids (RBuland and RPMH1) showed significant enhancement of endosperm proA (6.97-9.82 ppm), tryptophan (0.07-0.09%), and lysine (0.29-0.43%), while grain yield was at par with their original versions. The dissemination of reconstituted hybrids holds significant promise to alleviate vitamin-A deficiency and protein-energy malnutrition in developing countries.

Discovery of Novel Bacterial Chalcone Isomerases by a Sequence-Structure-Function-Evolution Strategy for Enzymatic Synthesis of (S)-Flavanones.

Chalcone isomerase (CHI) is a key enzyme in the biosynthesis of flavonoids in plants. The first bacterial CHI (CHIera ) was identified from Eubacterium ramulus, but its distribution, evolutionary source, substrate scope, and stereoselectivity are still unclear. Here, we describe the identification of 66 novel bacterial CHIs from Genbank using a novel Sequence-Structure-Function-Evolution (SSFE) strategy. These novel bacterial CHIs show diversity in substrate specificity towards various hydroxylated and methoxylated chalcones. The mutagenesis of CHIera according to the substrate binding models of these novel bacterial CHIs resulted in several variants with greatly improved activity towards these chalcones. Furthermore, the preparative scale conversion catalyzed by bacterial CHIs has been performed for five chalcones and revealed (S)-selectivity with up to 96 % ee, which provides an alternative biocatalytic route for the synthesis of (S)-flavanones in high yields.

Combining Metabolic and Monoterpene Synthase Engineering for de Novo Production of Monoterpene Alcohols in Escherichia coli.

The monoterpene alcohols acyclic nerol and bicyclic borneol are widely applied in the food, cosmetic, and pharmaceutical industries. The emerging synthetic biology enables microbial production to be a promising alternative for supplying monoterpene alcohols in an efficient and sustainable approach. In this study, we combined metabolic and plant monoterpene synthase engineering to improve the de novo production of nerol and borneol in prene-overproducing Escherichia coli. We engineered the growth-orthogonal neryl diphosphate (NPP) as the universal precursor of monoterpene alcohol biosynthesis and coexpressed nerol synthase (GmNES) from Glycine max to generate nerol or coexpressed the truncated bornyl diphosphate synthase (LdtBPPS) from Lippia dulcis for borneol production. Further, through site-directed mutation of LdtBPPS based on the structural simulation, we screened multiple variants that markedly elevated the production of acyclic nerol or bicyclic borneol, of which the LdtBPPSS488T mutant outperformed the wild-type LdtBPPS on borneol synthesis and the LdtBPPSF612A variant was superior to GmNES on nerol production. Subsequently, we overexpressed the endogenous Nudix hydrolase NudJ to facilitate the dephosphorylation of precursors and boosted the production of nerol and borneol from glucose. Finally, after the optimization of the fermentation process, the engineered strain ENO2 produced 966.55 mg/L nerol, and strain ENB57 generated 87.20 mg/L borneol in a shake flask, achieving the highest reported titers of nerol and borneol in microbes to date. This work shows a combinatorial engineering strategy for microbial production of natural terpene alcohols.

Lycopene ß-cyclase expression influences plant physiology, development, and metabolism in tobacco plants.

Carotenoids are important isoprenoids produced in the plastids of photosynthetic organisms that play key roles in photoprotection and antioxidative processes. ß-Carotene is generated from lycopene by lycopene ß-cyclase (LCYB). Previously, we demonstrated that the introduction of the Daucus carota (carrot) DcLCYB1 gene into tobacco (cv. Xanthi) resulted in increased levels of abscisic acid (ABA) and especially gibberellins (GAs), resulting in increased plant yield. In order to understand this phenomenon prior to exporting this genetic strategy to crops, we generated tobacco (Nicotiana tabacum cv. Petit Havana) mutants that exhibited a wide range of LCYB expression. Transplastomic plants expressing DcLCYB1 at high levels showed a wild-type-like growth, even though their pigment content was increased and their leaf GA1 content was reduced. RNA interference (RNAi) NtLCYB lines showed different reductions in NtLCYB transcript abundance, correlating with reduced pigment content and plant variegation. Photosynthesis (leaf absorptance, Fv/Fm, and light-saturated capacity of linear electron transport) and plant growth were impaired. Remarkably, drastic changes in phytohormone content also occurred in the RNAi lines. However, external application of phytohormones was not sufficient to rescue these phenotypes, suggesting that altered photosynthetic efficiency might be another important factor explaining their reduced biomass. These results show that LCYB expression influences plant biomass by different mechanisms and suggests thresholds for LCYB expression levels that might be beneficial or detrimental for plant growth.

Transforming yeast peroxisomes into microfactories for the efficient production of high-value isoprenoids.

Current approaches for the production of high-value compounds in microorganisms mostly use the cytosol as a general reaction vessel. However, competing pathways and metabolic cross-talk frequently prevent efficient synthesis of target compounds in the cytosol. Eukaryotic cells control the complexity of their metabolism by harnessing organelles to insulate biochemical pathways. Inspired by this concept, herein we transform yeast peroxisomes into microfactories for geranyl diphosphate-derived compounds, focusing on monoterpenoids, monoterpene indole alkaloids, and cannabinoids. We introduce a complete mevalonate pathway in the peroxisome to convert acetyl-CoA to several commercially important monoterpenes and achieve up to 125-fold increase over cytosolic production. Furthermore, peroxisomal production improves subsequent decoration by cytochrome P450s, supporting efficient conversion of (S)-(-)-limonene to the menthol precursor trans-isopiperitenol. We also establish synthesis of 8-hydroxygeraniol, the precursor of monoterpene indole alkaloids, and cannabigerolic acid, the cannabinoid precursor. Our findings establish peroxisomal engineering as an efficient strategy for the production of isoprenoids.

Transcriptional Analysis of Carotenoids Accumulation and Metabolism in a Pink-Fleshed Lemon Mutant.

Pink lemon is a spontaneous bud mutation of lemon (Citrus limon, L. Burm. f) characterized by the production of pink-fleshed fruits due to an unusual accumulation of lycopene. To elucidate the genetic determinism of the altered pigmentation, comparative carotenoid profiling and transcriptional analysis of both the genes involved in carotenoid precursors and metabolism, and the proteins related to carotenoid-sequestering structures were performed in pink-fleshed lemon and its wild-type. The carotenoid profile of pink lemon pulp is characterized by an increased accumulation of linear carotenoids, such as lycopene, phytoene and phytofluene, from the early stages of development, reaching their maximum in mature green fruits. The distinctive phenotype of pink lemon is associated with an up-regulation and down-regulation of the genes upstream and downstream the lycopene cyclase, respectively. In particular, 9-cis epoxycarotenoid dioxygenase genes were overexpressed in pink lemon compared with the wild-type, suggesting an altered regulation of abscisic acid biosynthesis. Similarly, during early development of the fruits, genes of the carotenoid-associated proteins heat shock protein 21, fibrillin 1 and 2 and orange gene were overexpressed in the pulp of the pink-fleshed lemon compared to the wild-type, indicating its increased capacity for sequestration, stabilization or accumulation of carotenes. Altogether, the results highlighted significant differences at the transcriptomic level between the pink-fleshed lemon and its wild-type, in terms of carotenoid metabolism and the capacity of stabilization in storage structures between the two accessions. Such changes may be either responsible for the altered carotenoid accumulation or in contrast, a metabolic consequence.