Multisite evaluation of phenotypic plasticity for specialized metabolites, some involved in carrot quality and disease resistance.

Renewed consumer demand motivates the nutritional and sensory quality improvement of fruits and vegetables. Specialized metabolites being largely involved in nutritional and sensory quality of carrot, a better knowledge of their phenotypic variability is required. A metabolomic approach was used to evaluate phenotypic plasticity level of carrot commercial varieties, over three years and a wide range of cropping environments spread over several geographical areas in France. Seven groups of metabolites have been quantified by HPLC or GC methods: sugars, carotenoids, terpenes, phenolic compounds, phenylpropanoids and polyacetylenes. A large variation in root metabolic profiles was observed, in relation with environment, variety and variety by environment interaction effects in decreasing order of importance. Our results show a clear diversity structuration based on metabolite content. Polyacetylenes, ß-pinene and α-carotene were identified mostly as relatively stable varietal markers, exhibiting static stability. Nevertheless, environment effect was substantial for a large part of carrot metabolic profile and various levels of phenotypic plasticity were observed depending on metabolites and varieties. A strong difference of environmental sensitivity between varieties was observed for several compounds, particularly myristicin, 6MM and D-germacrene, known to be involved in responses to biotic and abiotic stress. This work provides useful information about plasticity in the perspective of carrot breeding and production. A balance between constitutive content and environmental sensitivity for key metabolites should be reached for quality improvement in carrot and other vegetables.

Carotenoid gene expression explains the difference of carotenoid accumulation in carrot root tissues.

Main conclusion Variations in gene expression can partially explain the difference of carotenoid accumulation in secondary phloem and xylem of fleshy carrot roots. The carrot root is well divided into two different tissues separated by vascular cambium: the secondary phloem and xylem. The equilibrium between these two tissues represents an important issue for carrot quality, but the knowledge about the respective carotenoid accumulation is sparse. The aim of this work was (i) to investigate if variation in carotenoid biosynthesis gene expression could explain differences in carotenoid content in phloem and xylem tissues and (ii) to investigate if this regulation is differentially modulated in the respective tissues by water-restricted growing conditions. In this work, five carrot genotypes contrasting by their root color were studied in control and water-restricted conditions. Carotenoid content and the relative expression of 13 genes along the carotenoid biosynthesis pathway were measured in the respective tissues. Results showed that in orange genotypes and the purple one, carotenoid content was higher in phloem compared to xylem. For the red one, no differences were observed. Moreover, in control condition, variations in gene expression explained the different carotenoid accumulations in both tissues, while in water-restricted condition, no clear association between gene expression pattern and variations in carotenoid content could be detected except in orange-rooted genotypes. This work shows that the structural aspect of carrot root is more important for carotenoid accumulation in relation with gene expression levels than the consequences of expression changes upon water restriction.

Differential Pigment Accumulation in Carrot Leaves and Roots during Two Growing Periods.

Carotenoids are important secondary metabolites involved in plant growth and nutritional quality of vegetable crops. These pigments are highly accumulated in carrot root, but knowledge about the impact of environmental factors on their accumulation is limited. The purpose of this work was to investigate the impact of environmental variations on carotenoid accumulation in carrot leaves and roots. In this work, carrots were grown during two contrasting periods to maximize bioclimatic differences. In leaves, carotenoid and chlorophyll contents were lower in the less favorable growing conditions, whereas relative contents were well conserved for all genotypes, suggesting a common regulatory mechanism. The down-regulation of all genes under environmental constraints demonstrates that carotenoid accumulation is regulated at the transcriptional level. In roots, the decrease in α-carotene and lutein contents was accompanied by an increase of ß-carotene relative content. At the transcriptional level, LCYB and ZEP expression increased, whereas LCYE expression decreased, in the less favorable conditions, suggesting that carotenoid biosynthesis is switched toward the ß-branch.

Carotenoid content and root color of cultivated carrot: a candidate-gene association study using an original broad unstructured population.

Accumulated in large amounts in carrot, carotenoids are an important product quality attribute and therefore a major breeding trait. However, the knowledge of carotenoid accumulation genetic control in this root vegetable is still limited. In order to identify the genetic variants linked to this character, we performed an association mapping study with a candidate gene approach. We developed an original unstructured population with a broad genetic basis to avoid the pitfall of false positive detection due to population stratification. We genotyped 109 SNPs located in 17 candidate genes ­ mostly carotenoid biosynthesis genes ­ on 380 individuals, and tested the association with carotenoid contents and color components. Total carotenoids and ß-carotene contents were significantly associated with genes zeaxanthin epoxydase (ZEP), phytoene desaturase (PDS) and carotenoid isomerase (CRTISO) while α-carotene was associated with CRTISO and plastid terminal oxidase (PTOX) genes. Color components were associated most significantly with ZEP. Our results suggest the involvement of the couple PDS/PTOX and ZEP in carotenoid accumulation, as the result of the metabolic and catabolic activities respectively. This study brings new insights in the understanding of the carotenoid pathway in non-photosynthetic organs.

Functional gene polymorphism to reveal species history: the case of the CRTISO gene in cultivated carrots.

BACKGROUND: Carrot is a vegetable cultivated worldwide for the consumption of its root. Historical data indicate that root colour has been differentially selected over time and according to geographical areas. Root pigmentation depends on the relative proportion of different carotenoids for the white, yellow, orange and red types but only internally for the purple one. The genetic control for root carotenoid content might be partially associated with carotenoid biosynthetic genes. Carotenoid isomerase (CRTISO) has emerged as a regulatory step in the carotenoid biosynthesis pathway and could be a good candidate to show how a metabolic pathway gene reflects a species genetic history. METHODOLOGY/PRINCIPAL FINDINGS: In this study, the nucleotide polymorphism and the linkage disequilibrium among the complete CRTISO sequence, and the deviation from neutral expectation were analysed by considering population subdivision revealed with 17 microsatellite markers. A sample of 39 accessions, which represented different geographical origins and root colours, was used. Cultivated carrot was divided into two genetic groups: one from Middle East and Asia (Eastern group), and another one mainly from Europe (Western group). The Western and Eastern genetic groups were suggested to be differentially affected by selection: a signature of balancing selection was detected within the first group whereas the second one showed no selection. A focus on orange-rooted carrots revealed that cultivars cultivated in Asia were mainly assigned to the Western group but showed CRTISO haplotypes common to Eastern carrots. CONCLUSION: The carotenoid pathway CRTISO gene data proved to be complementary to neutral markers in order to bring critical insight in the cultivated carrot history. We confirmed the occurrence of two migration events since domestication. Our results showed a European background in material from Japan and Central Asia. While confirming the introduction of European carrots in Japanese resources, the history of Central Asia material remains unclear.

Differential selection on carotenoid biosynthesis genes as a function of gene position in the metabolic pathway: a study on the carrot and dicots.

BACKGROUND: Selection of genes involved in metabolic pathways could target them differently depending on the position of genes in the pathway and on their role in controlling metabolic fluxes. This hypothesis was tested in the carotenoid biosynthesis pathway using population genetics and phylogenetics. METHODOLOGY/PRINCIPAL FINDINGS: Evolutionary rates of seven genes distributed along the carotenoid biosynthesis pathway, IPI, PDS, CRTISO, LCYB, LCYE, CHXE and ZEP, were compared in seven dicot taxa. A survey of deviations from neutrality expectations at these genes was also undertaken in cultivated carrot (Daucus carota subsp. sativus), a species that has been intensely bred for carotenoid pattern diversification in its root during its cultivation history. Parts of sequences of these genes were obtained from 46 individuals representing a wide diversity of cultivated carrots. Downstream genes exhibited higher deviations from neutral expectations than upstream genes. Comparisons of synonymous and nonsynonymous substitution rates between genes among dicots revealed greater constraints on upstream genes than on downstream genes. An excess of intermediate frequency polymorphisms, high nucleotide diversity and/or high differentiation of CRTISO, LCYB1 and LCYE in cultivated carrot suggest that balancing selection may have targeted genes acting centrally in the pathway. CONCLUSIONS/SIGNIFICANCE: Our results are consistent with relaxed constraints on downstream genes and selection targeting the central enzymes of the carotenoid biosynthesis pathway during carrot breeding history.

Carotenoid biosynthesis genes provide evidence of geographical subdivision and extensive linkage disequilibrium in the carrot.

According to the history of the cultivated carrot, root colour can be considered as a structural factor of carrot germplasm. Therefore, molecular variations of carotenoid biosynthesis genes, these being involved in colour traits, represent a good putative source of polymorphism related to diversity structure. Seven candidate genes involved in the carotenoid biosynthesis pathway have been analysed from a sample of 48 individual plants, each one from a different cultivar of carrot (Daucus carota L. ssp. sativus). The cultivars were chosen to represent a large diversity and a wide range of root colour. A high single nucleotide polymorphism (SNP) frequency of 1 SNP per 22 bp (mean pi (sil) = 0.020) was found on average within these genes. The analysis of genetic structure from carotenoid biosynthesis gene sequences and 17 putatively neutral microsatellites showed moderate genetic differentiation between cultivars originating from the West and the East (F (ST) = 0.072), this being consistent with breeding history, but not previously evidenced by molecular tools. Surprisingly, carotenoid biosynthesis genes did not exhibit decay of LD (mean r (2) = 0.635) within the 700-1,000 bp analysed, even though a fast decay level of LD is expected in outcrossing species. The high level of intralocus LD found for carotenoid biosynthesis genes implies that candidate-gene association mapping for carrot root colour should be useful to validate gene function, but may be unable to identify precisely the causative variations involved in trait determinism. Finally this study affords the first molecular evidence of a genetic structure in cultivated carrot germplasm related to phylogeography.

Isolation and characterization of microsatellite markers from the phytopathogenic fungus Alternaria dauci.

Eleven polymorphic microsatellite markers were isolated from the necrotrophic phytopathogenic fungus Alternaria dauci based on enriched genomic libraries. In order to assess allelic variability, the microsatellite loci were analysed in a collection of 43 isolates. The number of detected alleles in 11 loci ranged from two to 24 (mean 10.4). Test of cross-species amplification and sequencing of the resulting amplicons showed that some of these microsatellites could be used in different species such as Alternaria solani, Alternaria bataticola and Alternaria zinniae.

Characterization of AT-rich microsatellites in common bean (Phaseolus vulgaris L.).

Polymorphism of microsatellite markers is often associated with the simple sequence repeat motif targeted. AT-rich microsatellites tend to be highly variable and this appears to be notable, especially in legume genomes. To analyze the value of AT-rich microsatellites for common bean (Phaseolus vulgaris L.), we developed a total of 85 new microsatellite markers, 74 of which targeted ATA or other AT-rich motif loci and 11 of which were made for GA, CA or CAC motif loci. We evaluated the loci for the level of allelic diversity in comparison to previously characterized microsatellites using a panel of 18 standard genotypes and genetically mapped any loci polymorphic in the DOR364 x G19833 population. The majority of the microsatellites produced single bands and detected single loci, however, 15 of the AT-rich microsatellites produced multiple or double banding patterns; while only one of the GA or CA-rich microsatellites did. The polymorphism information content (PIC) values averaged 0.892 and 0.600 for the AT and ATA motif microsatellites, respectively, but only 0.140 for the CA-rich microsatellites. GA microsatellites, which had a large average number of repeats, had high to intermediate PIC, averaging 0.706. A total of 45 loci could be genetically mapped and distribution of the loci across the genome was skewed towards non-distal locations with a greater prevalence of loci on linkage groups b02, b09 and b11. AT-rich microsatellites were found to be a useful source of polymorphic markers for mapping and diversity assessment in common bean that appears to uncover higher diversity than other types of simple sequence repeat markers.

Expression of carotenoid biosynthesis genes during carrot root development.

Carotenogenesis has been extensively studied in fruits and flower petals. Transcriptional regulation is thought to be the major factor in carotenoid accumulation in these organs. However, little is known about regulation in root organs. The root carotenoid content of carrot germplasm varies widely. The present study was conducted to investigate transcriptional regulation of carotenoid biosynthesis genes in relation to carotenoid accumulation during early carrot root development and up to 3 months after sowing. HPLC carotenoid content analysis and quantitative RT-PCR were compared to quantify the expression of eight genes encoding carotenoid biosynthesis enzymes during the development of white, yellow, orange, and red carrot roots. The genes chosen encode phytoene synthase (PSY1 and PSY2), phytoene desaturase (PDS), zeta-carotene desaturase (ZDS1 and ZDS2), lycopene epsilon-cyclase (LCYE), lycopene beta-cyclase (LCYB1), and zeaxanthin epoxidase (ZEP). All eight genes were expressed in the white cultivar even though it did not contain carotenoids. By contrast with fruit maturation, the expression of carotenogenic genes began during the early stages of development and then progressively increased for most of these genes during root development as the total carotenoid level increased in coloured carrots. The high expression of genes encoding LCYE and ZDS noted in yellow and red cultivars, respectively, might be consistent with the accumulation of lutein and lycopene, respectively. The results showed that the accumulation of total carotenoids during development and the accumulation of major carotenoids in the red and yellow cultivars might partially be explained by the transcriptional level of genes directing the carotenoid biosynthesis pathway.

A gene encoding a germin-like protein, identified by a cDNA-AFLP approach, is specifically expressed during germination of Phaseolus vulgaris.

In order to identify markers of germination in Phaseolus vulgaris L., a cDNA-amplified fragment length polymorphism (AFLP) approach was conducted on mRNAs from embryo axes and from cotyledons. Among changes observed throughout the germination process, a cDNA fragment not detected 9 h after imbibition (HAI) but present specifically in axes 24 HAI was further studied. The complete cDNA was recovered by rapid amplification of cDNA ends, then cloned and sequenced. It includes an open reading frame predicting a 206-amino-acid polypeptide of 21.8 kDa. Analysis of the nucleotide sequence and deduced amino acid sequence revealed a high homology with germin-like proteins (GLPs), and particularly with an auxin-binding protein from peach, ABP19, that belongs to the GLP family. Thus, we propose that this cDNA encodes the first GLP described in P. vulgaris, designated PvGLP1. Northern blot analysis carried out on mRNAs from seed axes showed a dramatic increase in PvGLP1 expression a few hours before radicle emergence (17 HAI). Among mature vegetative tissues, PvGLP1 expression was very weak in pods and not detected in leaves, stems or roots. Immunoblot analysis using antibodies raised against AtGER3 from Arabidopsis thaliana showed that the protein could be detected only in axes from the dry seed stage onwards, at a steady-state level. Then, PvGLP1 expression seems to be associated with the early stages of embryo axis growth. The high homology indicated with ABP19 led us to study the effect of different concentrations of indole-3-acetic acid (IAA) on PvGLP1 expression during germination. Whereas no effect was noticed at low concentrations (1, 5, 10 microM), a marked decrease in PvGLP1 mRNA level was observed in axes of seeds imbibed with 100 microM IAA. Thus, PvGLP1 gene expression is not stimulated by auxin and, moreover, it might be inhibited by high concentrations of IAA.

Molecular survey of genetic diversity in the endangered European mink Mustela lutreola.

The European mink Mustela lutreola is regarded as one of the most endangered mammals in the world. We chose to characterize microsatellite loci in order to investigate the pattern of decline of this species. We used primer pairs developed for a related species Mustela vison to genotype individual of Mustela lutreola. Out of 19 primer pairs used 8 were useful for our purpose. The conservation of primer sequence point out the problem of neutrality of some microsatellite loci as this conservation could be related to strong selection pressure on those loci. Finally we present the first data allowing an estimation of heterozygosity of French population of European mink.