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The regulation of flavonoid biosynthesis is only partially explored in pepper (Capsicum annuum L.). The genetic basis underlying flavonoid variation in pepper fruit was studied.Variation of flavonoids in fruit of a segregating F2 population was studied using LC–MS followed by quantitative trait locus (QTL) analysis. Near-isogenic lines (NILs), BC1S1 populations, virus-induced gene silenced (VIGS) and transgenic overexpression were used to confirm the QTL and the underlying candidate gene.A major QTL for flavonoid content was found in chromosome 5, and a CaMYB12-like transcription factor gene was identified as candidate gene. Near-isogenic lines (NILs) contrasting for CaMYB12-like confirmed its association with the flavonoid content variation. Virus-induced gene silencing (VIGS) of CaMYB12-like led to a significant decrease in the expression of several flavonoid pathway genes and a drastic decrease in flavonoid levels in silenced fruits. Expression of CaMYB12-like in the tomato slmyb12 mutant led to enhanced levels of several flavonoids in the fruit skin. Introgression of the CaMYB12-like allele into two cultivated varieties also increased flavonoid content in their fruits.A combination of metabolomic, genetic and gene functional analyses led to discovery of CaMYB12-like as a major regulator of flavonoid variation in pepper fruit and demonstrated its potential to breed for high-flavonoid content in cultivated pepper.

Anthocyanins are important pigments that impart color in plants. In Solanum, different species display various fruit or flower colors due to varying degrees of anthocyanin accumulation. Here we identified two anthocyanin-free mutants from an ethylmethane sulfonate-induced mutant library and naturally occurring mutants in Solanum melongena, with mutations in the 5′ splicing site of the second intron of dihydroflavonol-4-reductase (DFR) – leading to altered splicing. Further study revealed that alternative splicing of the second intron was closely related to anthocyanin accumulation in 17 accessions from three cultivated species: S. melongena, Solanum macrocarpon and Solanum aethiopicum, and their wild related species. Analysis of natural variations of DFR, using an expanded population including 282 accessions belonging to the spiny Solanum group, identified a single-nucleotide polymorphism in the MYB recognition site in the promoter region, which causes differential expression of DFR and affects anthocyanin accumulation in fruits of the detected accessions. Our study suggests that, owing to years of domestication, the natural variation in the DFR promoter region and the alternative splicing of the DFR gene account for altered anthocyanin accumulation during spiny Solanum domestication.

Contains fulltext : 198854.pdf (Publisher’s version ) (Open Access) BACKGROUND: Pollen development is central for plant reproduction and is assisted by changes of the transcriptome and proteome. At the same time, pollen development and viability is largely sensitive to stress, particularly to elevated temperatures. The transcriptomic and proteomic changes during pollen development and of different stages in response to elevated temperature was targeted to define the underlying molecular principles. RESULTS: The analysis of the transcriptome and proteome of Solanum lycopersicum pollen at tetrad, post-meiotic and mature stage before and after heat stress yielded a decline of the transcriptome but an increase of the proteome size throughout pollen development. Comparison of the transcriptome and proteome led to the discovery of two modes defined as direct and delayed translation. Here, genes of distinct functional processes are under the control of direct and delayed translation. The response of pollen to elevated temperature occurs rather at proteome, but not as drastic at the transcriptome level. Heat shock proteins, proteasome subunits, ribosomal proteins and eukaryotic initiation factors are most affected. On the example of heat shock proteins we demonstrate a decoupling of transcript and protein levels as well as a distinct regulation between the developmental stages. CONCLUSIONS: The transcriptome and proteome of developing pollen undergo drastic changes in composition and quantity. Changes at the proteome level are a result of two modes assigned as direct and delayed translation. The response of pollen to elevated temperature is mainly regulated at the proteome level, whereby proteins related to synthesis and degradation of proteins are most responsive and might play a central role in the heat stress response of pollen. 20 p.

Exploiting wild relatives of S. lycopersicum for quality traitsAna Marcela Víquez ZamoraTomatoes are consumed worldwide and became a model for crop plant research.A part of the research aims at expanding genetic diversity in tomato; this can be done by incorporating useful genes found in wild germplasm. In this thesis we focused on exploring the variation between commercial tomatoes and accessions of wild relatives. Especially, we focused on the speciesSolanumpimpinellifolium. We explored the genome and the metabolome of accessions ofS. pimpinellifoliumand several varieties ofS. lycopersicum. Finally a crossing population between the species was genetically analyzed. Clear differences were identified. We found genome regions related to differences between round and cherry tomatoes, TYLCV resistance, flavour and the phenylpropanoid pathway.S. pimpinellifoliumcertainly harbours useful genetic variability that can be (re-) introduced in tomato. Our results give an insight in the physical positions of metabolite related QTLs that can be used by breeders to exploitS. pimpinellifolium to improve tomato quality.

A diverse collection of 32 pepper accessions was analysed for variation in health-related metabolites, such as carotenoids, capsaicinoids, flavonoids and vitamins C and E. For each of the metabolites analysed, there was a lot of variation among the accessions and it was possible to identify accessions with high amounts of specific metabolites. While all accessions contained high levels of vitamin C, accession C. chinenseAC2212 was found to be an excellent source of vitamin E, whereas C. annuumLong Sweet accumulated high levels of the flavonoid quercetin. A genetical metabolomics approach was used to study the genetic basis of metabolic traits in a segregating F2 population based on a cross between two contrasting pepper genotypes. This led to the identification of several metabolites QTL hotspots. The genetic basis for the accumulation of several flavonoids in pepper fruit was further investigated, by combining metabolic, gene expression and candidate gene-based marker data. This provided valuable insight into the key genes important for flavonoid accumulation in pepper fruit. The results of this study will help breeders to assist future breeding programs aimed at optimizing the levels of nutritional compounds in pepper fruit.

This PhD project started with the composition of a diverse panel of genotypes that represented, (i) roughly the flavor variation in the commercial Capsicum annuum breeding program of Rijk Zwaan, (ii) parents of available mapping populations and (iii) some genotypes that were expected to have extraordinary flavors. The complete set consisted of 35 genotypes of which 24 genotypes were non-pungent. Volatile and non-volatile compounds as well as some breeding parameters were measured in mature fruits of all genotypes throughout the growing season. In addition, from three harvests the non-pungent genotypes were evaluated for taste by a trained descriptive sensory panel. The biochemical profiling with use of SPME-GC-MS allowed visualization of between- and within-species volatile compound variation. Principal components analysis (PCA) on the intensity patterns of 391 putative volatile compounds revealed individual grouping of C. chinense, C. baccatum var. pendulum and C. annuum, indicating potentially interesting volatile variation present in the former two groups. A large group of saturated and unsaturated esters were mainly responsible for the individual grouping of the C. chinense accessions. Due to the elevated acid concentrations and aberrant volatile profiles of the C. baccatum var. pendulum accessions PEN45 and PEN79, the two BIL populations derived from these accessions were identified as interesting candidates for further study. Compared to e.g. Mazurka the citrate concentration of the C. baccatum accessions was 2.5-3 times higher and the malate concentrations were even up to 12 times higher (Chapter 2). Based on the non-pungent genotypes, we found highly correlated clusters of volatiles and non-volatiles, which could be related to metabolic pathways and common biochemical precursors (Chapter 3). Contrasts between genotypes were caused by both qualitative and quantitative differences in these metabolic clusters, with the phenolic derivatives, higher alkanes, sesquiterpenes and lipid derived volatiles forming the major determinants. For the description of the non-pungent genotypes the panelists used fourteen attributes to describe the flavor sensation in the mouth/throat, which were the texture attributes crunchiness, stickiness of the skin, toughness and juiciness, the basic taste attributes sweetness and sourness and the retronasal flavor attributes aroma intensity, grassiness, green bean, carrot, fruity/apple, perfume, petrochemical and musty. The variation in flavor could be reduced into two major sensory contrasts, which were a texture related contrast and the basic sweet-sour contrast. The structure of the PCA plots resulting from the analysis with one harvest (Chapter 3) and the analysis with the combined three harvests (Chapter 4) remained almost identical, indicating the stability of these contrasts. To relate the sensory attributes to the metabolite data and to determine the importance of the individual compounds we used Random Forest regression on the individual harvests and on the three harvests together. Several predictors for the attributes aroma, fruity/apple, sourness and sweetness were found in common between harvests, which we proposed as key-metabolites involved in flavor determination of sweet pepper (Chapter 4). This list contains compounds with known relations to attributes, like sweetness and sugars, but also several compounds with new relations. In this respect we have demonstrated for the first time, that the metabolites p-menth-1-en-9-al, (E)-β-ocimene, (Z)-2-penten-1-ol, and 1-methyl-1,4-cyclohexadiene are related to fruity/apple taste and/or sweetness of pepper. For sourness the only compound with a consistent significant contribution was an unknown C6H8O2 compound. We postulated therefore the hypothesis that in pepper the effect of sourness related metabolites is masked by other volatile and non-volatile compounds or texture differences (Chapter 3). Subsequently in Chapter 4 we described a clear sweetness-sourness interaction and demonstrated that the masking effect of fructose and other sugars explained why we did not find organic acids contributing to the prediction of sourness. The major sensory attributes were also predicted between harvests. The Random Forest predictions of the texture related attributes (juiciness, toughness, crunchiness and stickiness of the skin) and sweetness were very good. The predictions of the attributes aroma intensity, sourness and fruity/apple were somewhat lower and more variable between harvests, especially in the second harvest. In general, we concluded that prediction of attributes with higher heritabilities works better and is more consistent over harvests (Chapter 4). Based on the results of the initial experiments (Chapter 2) the species C. baccatum was chosen for further study. To exploit the potential flavor wealth of C. baccatum PEN45 we combined interspecific crossing with embryo rescue, resulting in a multi-parent BC2S1 population, that was characterized for sensory and biochemical variation (Chapter 5). We developed a population specific genetic linkage map for QTL mapping of characterized traits. Because of the complex structure of our BC2S1 mapping population we encountered several limitations, such as accidental co-segregation, underrepresentation of color linked markers and pre-selection leading to skewness, which might have resulted in false positive or missed QTLs. Despite these limitations, we were still fairly well able to map several biochemical, physical and sensory traits, as demonstrated at first for the (monogenic) control traits red color and pungency in the BC2S1 mapping population and in second instance by validation of genetic effects via an experiment with near-isogenic lines (NILs).This two-step approach turned out to be very powerful, since it led to the identification of the main results from this thesis: (i) Asmall C. baccatum LG3 introgression causing an extraordinary effect on flavor, which resulted in significantly higher scores for the attributes aroma, flowers, spices, celery and chives. In an attempt to identify the responsible biochemical compounds few consistently up- and down-regulated metabolites were detected, including the well-known pepper compound 2-isobutyl-3-methoxypyrazine (down) and 6-methyl-4-oxo-5-heptenal (up); (ii) Two introgressions (LG10.1 and LG1) had major effects on terpenoid content of mature fruits, affecting at least fifteen different monoterpenes; (iii) A second LG3 fragment resulted in a strong increase in Brix (total soluble solids) without negative effects on fruit size (Chapter 5). In Chapter 6 some extra sensory results of the pungent genotypes are given and a comparison between the two C. baccatum pendulum BILs (PEN45 and PEN79 derived) is made in light of the overall results. Finally the perspectives for breeding are discussed and presented in the form of a flowchart for flavor improvement.

Planten beschikken over een enorme capaciteit om een breed scala aan secundaire metabolieten te produceren waarmee ze kunnen reageren op hun continu veranderende omgeving. Flavonoïden, een van de meest voorkomende soort secundaire metabolieten, zijn laagmoleculaire stoffen die van nature in vrijwel alle planten voorkomen. Ze zijn bij uiteenlopende natuurlijke processen betrokken. Zo ontstaat bijvoorbeeld de rode, blauwe en paarse kleur van veel bloemen door de aanwezigheid van anthocyanen, een specifieke klasse van flavonoïdpigmenten. Ook rijpe vruchten danken hun kleur vaak aan deze klasse van flavonoïden. Daarnaast spelen flavonoïden een rol bij processen zoals bescherming van planten tegen schadelijke UV-straling, afweer tegen infecties, pollenvorming en fertiliteit. Omdat flavonoïden wijdverspreid voorkomen in het plantenrijk, vormen deze stoffen een permanent onderdeel van ons plantaardig voedsel. Een deel van de gezondheidsbevorderende effecten van groenten en fruit wordt toegeschreven aan de aanwezigheid van bepaalde flavonoïden. Hoewel flavonoïden in vrijwel alle planten voorkomen, zijn sommige klassen specifiek voor een bepaalde plantensoort, terwijl deze ondervertegenwoordigd of geheel afwezig kunnen zijn in andere plantensoorten. Vanuit dit oogpunt kan het wenselijk zijn om te selecteren voor gewassen met bepaalde (hoeveelheden van) flavonoïden, dan wel de samenstelling daarvan aan te passen. Onderzoek naar de mogelijkheden om de productie van flavonoïden in planten te veranderen wordt veelal uitgevoerd om de aantrekkelijkheid van sierteeltgewassen óf de voedingswaarde van bepaalde gewassen te verhogen. Elio Schijlen onderzocht de genetische modificatie van de biosyntheseroute van flavonoïden in tomaat. Hiermee wil hij meer inzicht geven in de endogene flavonoïdbiosynthese én de mogelijkheden onderzoeken om nieuwe flavonoïden te produceren die van nature niet voorkomen in tomaten.