A desert Chlorella sp. that thrives at extreme high-light intensities using a unique photoinhibition protection mechanism.

While light is the driving force of photosynthesis, excessive light can be harmful. Photoinhibition is one of the key processes that limit photosynthetic productivity. A well-defined mechanism that protects from photoinhibition has been described. Chlorella ohadii is a green micro-alga, isolated from biological desert soil crusts, which thrives under extreme high light (HL). Here, we show that this alga evolved unique protection mechanisms distinct from those of the green alga Chlamydomonas reinhardtii or plants. When grown under extreme HL, a drastic reduction in the size of light harvesting antennae occurs, resulting in the presence of core photosystem II, devoid of outer and inner antennas. This is accompanied by a massive accumulation of protective carotenoids and proteins that scavenge harmful radicals. At the same time, several elements central to photoinhibition protection in C. reinhardtii, such as psbS, light harvesting complex stress-related, photosystem II protein phosphorylation and state transitions are entirely absent or were barely detected. In addition, a carotenoid biosynthesis-related protein accumulates in the thylakoid membranes of HL cells and may function in sensing HL and protecting the cell from photoinhibition. Taken together, a unique photoinhibition protection mechanism evolved in C. ohadii, enabling the species to thrive under extreme-light intensities where other photosynthetic organisms fail to survive.

Pan-genome and multi-parental framework for high-resolution trait dissection in melon (Cucumis melo).

Linking genotype with phenotype is a fundamental goal in biology and requires robust data for both. Recent advances in plant-genome sequencing have expedited comparisons among multiple-related individuals. The abundance of structural genomic within-species variation that has been discovered indicates that a single reference genome cannot represent the complete sequence diversity of a species, leading to the expansion of the pan-genome concept. For high-resolution forward genetics, this unprecedented access to genomic variation should be paralleled and integrated with phenotypic characterization of genetic diversity. We developed a multi-parental framework for trait dissection in melon (Cucumis melo), leveraging a novel pan-genome constructed for this highly variable cucurbit crop. A core subset of 25 diverse founders (MelonCore25), consisting of 24 accessions from the two widely cultivated subspecies of C. melo, encompassing 12 horticultural groups, and 1 feral accession was sequenced using a combination of short- and long-read technologies, and their genomes were assembled de novo. The construction of this melon pan-genome exposed substantial variation in genome size and structure, including detection of ~300 000 structural variants and ~9 million SNPs. A half-diallel derived set of 300 F2 populations, representing all possible MelonCore25 parental combinations, was constructed as a framework for trait dissection through integration with the pan-genome. We demonstrate the potential of this unified framework for genetic analysis of various melon traits, including rind color intensity and pattern, fruit sugar content, and resistance to fungal diseases. We anticipate that utilization of this integrated resource will enhance genetic dissection of important traits and accelerate melon breeding.

Underground heterosis for yield improvement in melon.

Heterosis, the superiority of hybrids over their parents, is a major genetic force associated with plant fitness and crop yield enhancement. We investigated root-mediated yield heterosis in melons (Cucumis melo) by characterizing a common variety grafted onto 190 hybrid rootstocks, resulting from crossing 20 diverse inbreds in a diallel-mating scheme. Hybrid rootstocks improved yield by more than 40% compared with their parents, and the best hybrid yield outperformed the reference commercial variety by 65% under both optimal and minimal irrigation treatments. To characterize the genetics of underground heterosis we conducted whole genome re-sequencing of the 20 founder lines, and showed that parental genetic distance was no predictor for the level of heterosis. Through inference of the 190 hybrid genotypes from their parental genomes, followed by genome-wide association analysis, we mapped multiple quantitative trait loci for root-mediated yield. Yield enhancement of the four best-performing hybrid rootstocks was validated in multiple experiments with four different scion varieties. Our grafting approach is complementary to the common roots genetic approach that focuses mainly on variation in root system architecture, and is a step towards discovery of candidate genes involved in root function and yield enhancement.

Targeted mutagenesis of two homologous ATP-binding cassette subfamily G (ABCG) genes in tomato confers resistance to parasitic weed Phelipanche aegyptiaca.

Phelipanche aegyptiaca and Orobanche spp. are obligate plant root-parasitic weeds that cause extensive damage in agricultural crop plants. Their germination requires exposure to strigolactones (SLs) exuded by the host plant roots. Here we studied genes in the host plant tomato involved in SL exudation and their impact on parasitic weeds. We provide evidence that CRISPR/Cas9-mediated targeted mutagenesis of two homologous ATP-binding cassette subfamily G (ABCG) genes, ABCG44 (Solyc08g067610) and ABCG45 (Solyc08g067620), in tomato significantly reduces SLs in the root exudate and abolishes germination of the root-parasitic weed P. aegyptiaca. Based on genome sequence similarity between ABCG44 and ABCG45, a 20-bp target sequence in their exon region was selected to design single guide RNA targeting both genes using CRISPR/Cas9. The plant binary vector constructs harboring the specific Cas9 and single guide RNA were transformed into tomato. Selected T0 mutated tomato plants showed different types of deletions at both gene loci. Genotype analysis of T1 plants suggested stable inheritance of the introduced mutations without any potential off-target effects. The phenotype of Cas9-mutated plants included increased shoot branching and growth of axillary buds, and reduced length of primary stems. Interestingly, reduced germination of P. aegyptiaca resulted from a decrease in the SL orobanchol in the root exudate of Cas9-mutated plants; however, orobanchol content in the root extract was unchanged compared to control plants. Moreover, in single and double ABCG mutants, expression of the SL-biosynthesis genes CCD8 and MAX1 decreased. The current study offers insights into CRISPR-mediated mutagenesis of ABCG genes, which could serve as an efficient control method to prevent root-parasitic weed germination.

The desert green algae Chlorella ohadii thrives at excessively high light intensities by exceptionally enhancing the mechanisms that protect photosynthesis from photoinhibition.

Although light is the driving force of photosynthesis, excessive light can be harmful. One of the main processes that limits photosynthesis is photoinhibition, the process of light-induced photodamage. When the absorbed light exceeds the amount that is dissipated by photosynthetic electron flow and other processes, damaging radicals are formed that mostly inactivate photosystem II (PSII). Damaged PSII must be replaced by a newly repaired complex in order to preserve full photosynthetic activity. Chlorella ohadii is a green microalga, isolated from biological desert soil crusts, that thrives under extreme high light and is highly resistant to photoinhibition. Therefore, C. ohadii is an ideal model for studying the molecular mechanisms underlying protection against photoinhibition. Comparison of the thylakoids of C. ohadii cells that were grown under low light versus extreme high light intensities found that the alga employs all three known photoinhibition protection mechanisms: (i) massive reduction of the PSII antenna size; (ii) accumulation of protective carotenoids; and (iii) very rapid repair of photodamaged reaction center proteins. This work elucidated the molecular mechanisms of photoinhibition resistance in one of the most light-tolerant photosynthetic organisms, and shows how photoinhibition protection mechanisms evolved to marginal conditions, enabling photosynthesis-dependent life in severe habitats.

High-density NGS-based map construction and genetic dissection of fruit shape and rind netting in Cucumis melo.

Melon is an important crop that exhibits broad variation for fruit morphology traits that are the substrate for genetic mapping efforts. In the post-genomic era, the link between genetic maps and physical genome assemblies is key for leveraging QTL mapping results for gene cloning and breeding purposes. Here, using a population of 164 melon recombinant inbred lines (RILs) that were subjected to genotyping-by-sequencing, we constructed and compared high-density sequence- and linkage-based recombination maps that were aligned to the reference melon genome. These analyses reveal the genome-wide variation in recombination frequency and highlight regions of disrupted collinearity between our population and the reference genome. The population was phenotyped over 3 years for fruit size and shape as well as rind netting. Four QTLs were detected for fruit size, and they act in an additive manner, while significant epistatic interaction was found between two neutral loci for this trait. Fruit shape displayed transgressive segregation that was explained by the action of four QTLs, contributed by alleles from both parents. The complexity of rind netting was demonstrated on a collection of 177 diverse accessions. Further dissection of netting in our RILs population, which is derived from a cross of smooth and densely netted parents, confirmed the intricacy of this trait and the involvement of major locus and several other interacting QTLs. A major netting QTL on chromosome 2 co-localized with results from two additional populations, paving the way for future study toward identification of a causative gene for this trait.

The Role of Carotenogenic Metabolic Flux in Carotenoid Accumulation and Chromoplast Differentiation: Lessons From the Melon Fruit.

Carotenoids have various roles in plant physiology. Plant carotenoids are synthesized in plastids and are highly abundant in the chromoplasts of ripening fleshy fruits. Considerable research efforts have been devoted to elucidating mechanisms that regulate carotenoid biosynthesis, yet, little is known about the mechanism that triggers storage capacity, mainly through chromoplast differentiation. The Orange gene (OR) product stabilizes phytoene synthase protein (PSY) and triggers chromoplast differentiation. OR underlies carotenoid accumulation in orange cauliflower and melon. The OR's 'golden SNP', found in melon, alters the highly evolutionary conserved Arginine108 to Histidine and controls ß-carotene accumulation in melon fruit, in a mechanism yet to be elucidated. We have recently shown that similar carotenogenic metabolic flux is active in non-orange and orange melon fruit. This flux probably leads to carotenoid turnover but known carotenoid turnover products are not detected in non-orange fruit. Arrest of this metabolic flux, using chemical inhibitors or mutations, induces carotenoid accumulation and biogenesis of chromoplasts, regardless of the allelic state of OR. We suggest that the 'golden SNP' induces ß-carotene accumulation probably by negatively affecting the capacity to synthesize downstream compounds. The accumulation of carotenoids induces chromoplast biogenesis through a metabolite-induced mechanism. Carotenogenic turnover flux can occur in non-photosynthetic tissues, which do not accumulate carotenoids. Arrest of this flux by the 'golden SNP' or other flux-arrest mutations is a potential tool for the biofortification of agricultural products with carotenoids.

The multi-allelic APRR2 gene is associated with fruit pigment accumulation in melon and watermelon.

Color and pigment contents are important aspects of fruit quality and consumer acceptance of cucurbit crops. Here, we describe the independent mapping and cloning of a common causative APRR2 gene regulating pigment accumulation in melon and watermelon. We initially show that the APRR2 transcription factor is causative for the qualitative difference between dark and light green rind in both crops. Further analyses establish the link between sequence or expression level variations in the CmAPRR2 gene and pigment content in the rind and flesh of mature melon fruits. A genome-wide association study (GWAS) of young fruit rind color in a panel composed of 177 diverse melon accessions did not result in any significant association, leading to an earlier assumption that multiple genes are involved in shaping the overall phenotypic variation in this trait. Through resequencing of 25 representative accessions and allelism tests between light rind accessions, we show that multiple independent single nucleotide polymorphisms in the CmAPRR2 gene are causative of the light rind phenotype. The multi-haplotypic nature of this gene explains the lack of detection power obtained through genotyping by sequencing-based GWAS and confirms the pivotal role of this gene in shaping fruit color variation in melon. This study demonstrates the power of combining bi- and multi-allelic designs with deep sequencing, to resolve lack of power due to high haplotypic diversity and low allele frequencies. Due to its central role and broad effect on pigment accumulation in fruits, the APRR2 gene is an attractive target for carotenoid bio-fortification of cucurbit crops.

Deciphering genetic factors that determine melon fruit-quality traits using RNA-Seq-based high-resolution QTL and eQTL mapping.

Combined quantitative trait loci (QTL) and expression-QTL (eQTL) mapping analysis was performed to identify genetic factors affecting melon (Cucumis melo) fruit quality, by linking genotypic, metabolic and transcriptomic data from a melon recombinant inbred line (RIL) population. RNA sequencing (RNA-Seq) of fruit from 96 RILs yielded a highly saturated collection of > 58 000 single-nucleotide polymorphisms, identifying 6636 recombination events that separated the genome into 3663 genomic bins. Bin-based QTL analysis of 79 RILs and 129 fruit-quality traits affecting taste, aroma and color resulted in the mapping of 241 QTL. Thiol acyltransferase (CmThAT1) gene was identified within the QTL interval of its product, S-methyl-thioacetate, a key component of melon fruit aroma. Metabolic activity of CmThAT1-encoded protein was validated in bacteria and in vitro. QTL analysis of flesh color intensity identified a candidate white-flesh gene (CmPPR1), one of two major loci determining fruit flesh color in melon. CmPPR1 encodes a member of the pentatricopeptide protein family, involved in processing of RNA in plastids, where carotenoid and chlorophyll pigments accumulate. Network analysis of > 12 000 eQTL mapped for > 8000 differentially expressed fruit genes supported the role of CmPPR1 in determining the expression level of plastid targeted genes. We highlight the potential of RNA-Seq-based QTL analysis of small to moderate size, advanced RIL populations for precise marker-assisted breeding and gene discovery. We provide the following resources: a RIL population genotyped with a unique set of SNP markers, confined genomic segments that harbor QTL governing 129 traits and a saturated set of melon eQTLs.

Genome-Wide Linkage-Disequilibrium Mapping to the Candidate Gene Level in Melon (Cucumis melo).

Cucumis melo is highly diverse for fruit traits providing wide breeding and genetic research opportunities, including genome-wide association (GWA) analysis. We used a collection of 177 accessions representing the two C. melo subspecies and 11 horticultural groups for detailed characterization of fruit traits variation and evaluation of the potential of GWA for trait mapping in melon. Through genotyping-by-sequencing, 23,931 informative SNPs were selected for genome-wide analyses. We found that linkage-disequilibrium decays at ~100 Kb in this collection and that population structure effect on association results varies between traits. We mapped several monogenic traits to narrow intervals overlapping with known causative genes, demonstrating the potential of diverse collections and GWA for mapping Mendelian traits to a candidate-gene level in melon. We further report on mapping of fruit shape quantitative trait loci (QTLs) and comparison with multiple previous QTL studies. Expansion of sample size and a more balanced representation of taxonomic groups might improve efficiency for simple traits dissection. But, as in other plant species, integrated linkage-association multi-allelic approaches are likely to produce better combination of statistical power, diversity capture and mapping resolution in melon. Our data can be utilized for selection of the most appropriate accessions for such approaches.

Distinct Mechanisms of the ORANGE Protein in Controlling Carotenoid Flux.

ß-Carotene adds nutritious value and determines the color of many fruits, including melon (Cucumis melo). In melon mesocarp, ß-carotene accumulation is governed by the Orange gene (CmOr) golden single-nucleotide polymorphism (SNP) through a yet to be discovered mechanism. In Arabidopsis (Arabidopsis thaliana), OR increases carotenoid levels by posttranscriptionally regulating phytoene synthase (PSY). Here, we identified a CmOr nonsense mutation (Cmor-lowß) that lowered fruit ß-carotene levels with impaired chromoplast biogenesis. Cmor-lowß exerted a minimal effect on PSY transcripts but dramatically decreased PSY protein levels and enzymatic activity, leading to reduced carotenoid metabolic flux and accumulation. However, the golden SNP was discovered to not affect PSY protein levels and carotenoid metabolic flux in melon fruit, as shown by carotenoid and immunoblot analyses of selected melon genotypes and by using chemical pathway inhibitors. The high ß-carotene accumulation in golden SNP melons was found to be due to a reduced further metabolism of ß-carotene. This was revealed by genetic studies with double mutants including carotenoid isomerase (yofi), a carotenoid-isomerase nonsense mutant, which arrests the turnover of prolycopene. The yofi F2 segregants accumulated prolycopene independently of the golden SNP Moreover, Cmor-lowß was found to inhibit chromoplast formation and chloroplast disintegration in fruits from 30 d after anthesis until ripening, suggesting that CmOr regulates the chloroplast-to-chromoplast transition. Taken together, our results demonstrate that CmOr is required to achieve PSY protein levels to maintain carotenoid biosynthesis metabolic flux but that the mechanism of the CmOr golden SNP involves an inhibited metabolism downstream of ß-carotene to dramatically affect both carotenoid content and plastid fate.

A bulk segregant transcriptome analysis reveals metabolic and cellular processes associated with Orange allelic variation and fruit ß-carotene accumulation in melon fruit.

BACKGROUND: Melon fruit flesh color is primarily controlled by the "golden" single nucleotide polymorhism of the "Orange" gene, CmOr, which dominantly triggers the accumulation of the pro-vitamin A molecule, ß-carotene, in the fruit mesocarp. The mechanism by which CmOr operates is not fully understood. To identify cellular and metabolic processes associated with CmOr allelic variation, we compared the transcriptome of bulks of developing fruit of homozygous orange and green fruited F3 families derived from a cross between orange and green fruited parental lines. RESULTS: Pooling together F3 families that share same fruit flesh color and thus the same CmOr allelic variation, normalized traits unrelated to CmOr allelic variation. RNA sequencing analysis of these bulks enabled the identification of differentially expressed genes. These genes were clustered into functional groups. The relatively enriched functional groups were those involved in photosynthesis, RNA and protein regulation, and response to stress. CONCLUSIONS: The differentially expressed genes and the enriched processes identified here by bulk segregant RNA sequencing analysis are likely part of the regulatory network of CmOr. Our study demonstrates the resolution power of bulk segregant RNA sequencing in identifying genes related to commercially important traits and provides a useful tool for better understanding the mode of action of CmOr gene in the mediation of carotenoid accumulation.

A Kelch Domain-Containing F-Box Coding Gene Negatively Regulates Flavonoid Accumulation in Muskmelon.

The flavonoids are phenylpropanoid-derived metabolites that are ubiquitous in plants, playing many roles in growth and development. Recently, we observed that fruit rinds of yellow casaba muskmelons (Cucumis melo 'Inodorous Group') accumulate naringenin chalcone, a yellow flavonoid pigment. With RNA-sequencing analysis of bulked segregants representing the tails of a population segregating for naringenin chalcone accumulation followed by fine mapping and genetic transformation, we identified a Kelch domain-containing F-box protein coding (CmKFB) gene that, when expressed, negatively regulates naringenin chalcone accumulation. Additional metabolite analysis indicated that downstream flavonoids are accumulated together with naringenin chalcone, whereas CmKFB expression diverts the biochemical flux toward coumarins and general phenylpropanoids. These results show that CmKFB functions as a posttranscriptional regulator that diverts flavonoid metabolic flux.

Systems approach for exploring the intricate associations between sweetness, color and aroma in melon fruits.

BACKGROUND: Melon (Cucumis melo) fruits exhibit phenotypic diversity in several key quality determinants such as taste, color and aroma. Sucrose, carotenoids and volatiles are recognized as the key compounds shaping the above corresponding traits yet the full network of biochemical events underlying their synthesis have not been comprehensively described. To delineate the cellular processes shaping fruit quality phenotypes, a population of recombinant inbred lines (RIL) was used as a source of phenotypic and genotypic variations. In parallel, ripe fruits were analyzed for both the quantified level of 77 metabolic traits directly associated with fruit quality and for RNA-seq based expression profiles generated for 27,000 unigenes. First, we explored inter-metabolite association patterns; then, we described metabolites versus gene association patterns; finally, we used the correlation-based associations for predicting uncharacterized synthesis pathways. RESULTS: Based on metabolite versus metabolite and metabolite versus gene association patterns, we divided metabolites into two key groups: a group including ethylene and aroma determining volatiles whose accumulation patterns are correlated with the expression of genes involved in the glycolysis and TCA cycle pathways; and a group including sucrose and color determining carotenoids whose accumulation levels are correlated with the expression of genes associated with plastid formation. CONCLUSIONS: The study integrates multiple processes into a genome scale perspective of cellular activity. This lays a foundation for deciphering the role of gene markers associated with the determination of fruit quality traits.

A 'golden' SNP in CmOr governs the fruit flesh color of melon (Cucumis melo).

The flesh color of Cucumis melo (melon) is genetically determined, and can be white, light green or orange, with ß-carotene being the predominant pigment. We associated carotenoid accumulation in melon fruit flesh with polymorphism within CmOr, a homolog of the cauliflower BoOr gene, and identified CmOr as the previously described gf locus in melon. CmOr was found to co-segregate with fruit flesh color, and presented two haplotypes (alleles) in a broad germplasm collection, one being associated with orange flesh and the second being associated with either white or green flesh. Allelic variation of CmOr does not affect its transcription or protein level. The variation also does not affect its plastid subcellular localization. Among the identified single nucleotide polymorphisms (SNPs) between CmOr alleles in orange versus green/white-flesh fruit, a single SNP causes a change of an evolutionarily highly conserved arginine to histidine in the CmOr protein. Functional analysis of CmOr haplotypes in an Arabidopsis callus system confirmed the ability of the CmOr orange haplotype to induce ß-carotene accumulation. Site-directed mutagenesis of the CmOr green/white haplotype to change the CmOR arginine to histidine triggered ß-carotene accumulation. The identification of the 'golden' SNP in CmOr, which is responsible for the non-orange and orange melon fruit phenotypes, provides new tools for studying the Or mechanism of action, and suggests genome editing of the Or gene for nutritional biofortification of crops.

Recombinant yeast as a functional tool for understanding bitterness and cucurbitacin biosynthesis in watermelon (Citrullus spp.).

Cucurbitacins are a group of bitter-tasting oxygenated tetracyclic triterpenes that are produced in the family Cucurbitaceae and other plant families. The natural roles of cucurbitacins in plants are probably related to defence against pathogens and pests. Cucurbitadienol, a triterpene synthesized from oxidosqualene, is the first committed precursor to cucurbitacins produced by a specialized oxidosqualene cyclase termed cucurbitadienol synthase. We explored cucurbitacin accumulation in watermelon in relation to bitterness. Our findings show that cucurbitacins are accumulated in bitter-tasting watermelon, Citrullus lanatus var. citroides, as well as in their wild ancestor, C. colocynthis, but not in non-bitter commercial cultivars of sweet watermelon (C. lanatus var. lanatus). Molecular analysis of genes expressed in the roots of several watermelon accessions led to the isolation of three sequences (CcCDS1, CcCDS2 and ClCDS1), all displaying high similarity to the pumpkin CpCPQ, encoding a protein previously shown to possess cucurbitadienol synthase activity. We utilized the Saccharomyces cerevisiae strain BY4743, heterozygous for lanosterol synthase, to probe for possible encoded cucurbitadienol synthase activity of the expressed watermelon sequences. Functional expression of the two sequences isolated from C. colocynthis (CcCDS1 and CcCDS2) in yeast revealed that only CcCDS2 possessed cucurbitadienol synthase activity, while CcCDS1 did not display cucurbitadienol synthase activity in recombinant yeast. ClCDS1 isolated from C. lanatus var. lanatus is almost identical to CcCDS1. Our results imply that CcCDS2 plays a role in imparting bitterness to watermelon. Yeast has been an excellent diagnostic tool to determine the first committed step of cucurbitacin biosynthesis in watermelon.

Formation of norisoprenoid flavor compounds in carrot (Daucus carota L.) roots: characterization of a cyclic-specific carotenoid cleavage dioxygenase 1 gene.

Carotenoids are isoprenoid pigments that upon oxidative cleavage lead to the production of norisoprenoids that have profound effect on flavor and aromas of agricultural products. The biosynthetic pathway to norisoprenoids in carrots (Daucus carota L.) is still largely unknown. We found the volatile norisoprenoids farnesylacetone, α-ionone, and ß-ionone accumulated in Nairobi, Rothild, and Purple Haze cultivars but not in Yellowstone and Creme de Lite in a pattern reflecting their carotenoid content. A cDNA encoding a protein with carotenoid cleavage dioxygenase activity, DcCCD1, was identified in carrot and was overexpressed in Escherichia coli strains previously engineered to produce different carotenoids. The recombinant DcCCD1 enzyme cleaves cyclic carotenes to generate α- and ß-ionone. No cleavage products were found when DcCCD1 was co-expressed in E. coli strains accumulating non-cyclic carotenoids, such as phytoene or lycopene. Our results suggest a role for DcCCD1 in carrot flavor biosynthesis.

Genetic and chemical characterization of an EMS induced mutation in Cucumis melo CRTISO gene.

In order to broaden the available genetic variation of melon, we developed an ethyl methanesulfonate mutation library in an orange-flesh 'Charentais' type melon line that accumulates ß-carotene. One mutagenized M2 family segregated for a novel recessive trait, a yellow-orange fruit flesh ('yofI'). HPLC analysis revealed that 'yofI' accumulates pro-lycopene (tetra-cis-lycopene) as its major fruit pigment. The altered carotenoid composition of 'yofI' is associated with a significant change of the fruit aroma since cleavage of ß-carotene yields different apocarotenoids than the cleavage of pro-lycopene. Normally, pro-lycopene is further isomerized by CRTISO (carotenoid isomerase) to yield all-trans-lycopene, which is further cyclized to ß-carotene in melon fruit. Cloning and sequencing of 'yofI' CRTISO identified two mRNA sequences which lead to truncated forms of CRTISO. Sequencing of the genomic CRTISO identified an A-T transversion in 'yofI' which leads to a premature STOP codon. The early carotenoid pathway genes were up regulated in yofI fruit causing accumulation of other intermediates such as phytoene and ζ-carotene. Total carotenoid levels are only slightly increased in the mutant. Mutants accumulating pro-lycopene have been reported in both tomato and watermelon fruits, however, this is the first report of a non-lycopene accumulating fruit showing this phenomenon.

Induced mutation in ß-CAROTENE HYDROXYLASE results in accumulation of ß-carotene and conversion of red to orange color in pepper fruit.

Pepper fruit is typically red, but green, orange and yellow cultivars are gaining consumer acceptance. This color variation is mainly due to variations in carotenoid composition. Orange color in pepper can result from a number of carotenoid profiles, but its genetic basis is only partly known. We identified an EMS-induced orange-fruited mutant using the wild-type blocky red-fruited cultivar 'Maor' as progenitor. This mutant accumulates mainly ß-carotene in its fruit, instead of the complex pattern of red and yellow carotenoids in 'Maor'. We identified an A(709) to G transition in the cDNA of ß-CAROTENE HYDROXYLASE2 in the orange pepper and complete co-segregation of this single-nucleotide polymorphism with the mutated phenotype. We therefore hypothesized that ß-CAROTENE HYDROXYLASE2 controls the orange mutation in pepper. Interestingly, the expression of ß-CAROTENE HYDROXYLASE2 and additional carotenogenesis genes was elevated in the orange fruit compared with the red fruit, indicating possible feedback regulation of genes in the pathway. Because carotenoids serve as precursors for volatile compounds, we compared the volatile profiles of the two parents. The orange pepper contained more volatile compounds than 'Maor', with predominant elevation of norisoprenoids derived from ß-carotene degradation, while sesquiterpenes predominated in the red fruit. Because of the importance of ß-carotene as a provitamin A precursor in the human diet, the orange-fruited mutant might serve as a natural source for pepper fruit biofortification. Moreover, the change in volatile profile may result in a fruit flavor that differs from other pepper cultivars.