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Flavonoids form a large class of secondary metabolites with numerous functions in plants, such as conferring disease resistance, coping with abiotic stress and attracting pollinators and seed dispersers. More than 5000 different flavonoids have been identified in the plant kingdom and they can be classified in several classes based on their structure, such chalcones, flavonols and anthocyanins. The latter group consists of red to purple pigments found in fruits like apple, strawberry and vegetables like red cabbage. Because of colour research in flowers such as petunia, the flavonoid pathway is one of the best studied metabolic pathways in plants and most of the structural genes and enzymes of the flavonoid pathway are known. A schematic of the flavonoid pathway is shown in figure 4. The first flavonoid is the chalcone naringenin chalcone, which is produced by the action of chalcone synthase (CHS). Through several enzymatic conversions the downstream flavonoids are made. Depending on the presence and activity of the different pathway genes, different plant species produce different types of flavonoids in their tissues. In tomato fruit peel, the major flavonoids which accumulate are naringenin chalcone and the flavonol quercetin, depicted by red triangles. The red arrows show the pathway steps active in tomato fruit.
Figure 4. Click to enlarge. Schematic of the flavonoid pathway in plants. The anthocyanins are depicted in colour. Red arrows mark the main flavonoids in tomato fruit peel: naringenin chalcone and quercetin.
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The structural genes in the flavonoid pathway of tomato are known, based on their homology with the orthologs of Petunia. The cDNA sequences of the main flavonoid genes expressed in tomato fruit are provided as fasta files in worksheet Flavonoid genes. A fasta file always starts with >, followed by the name of the sequence. As you can see, this worksheet contains the sequences of 7 flavonoid genes, acting from the beginning of the pathway (CHS) until the flavonols (FLS). Note that for some gene multiple copies are present in the genome. In order to see if any of the flavonoid structural genes is located in the IL1B QTL region, you need to find the genomic position of the tomato flavonoid genes, by BLASTING these cDNA sequences at SGN (https://solgenomics.net/) against the chromosomes of tomato,release SL2.40 of the tomato genome. Go to Tools, BLAST. At Categories select Tomato Genome (other datasets); at Database select WGS Chromosomes (SL2.40), at Program select blastn; at Query select autodetect. Copy column 1 of worksheet Flavonoid genes into the query form and run the BLAST analysis. From the output, extract the chromosome and approximate position of the structural flavonoid genes and fill this in in Table 1. Note: make sure to select the position at which the whole cDNA sequence is covered in the BLAST!
Table 1. Click to enlarge. Chromosome and position of the flavonoid genes tested
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- Describe how the expression of these genes develops during ripening in WT and IL1B fruits.
- Can these results explain the difference in flavonoid content, especially naringenin chalcone, in the peel of WT and IL1B tomato fruits? Hint: take into account the high expression of CHS vs the low expression of CHI.
- What do these results suggest with respect to the type of gene that may cause this phenotype?
Figure 6. Click to enlarge. Expression of flavonoid genes in ripening fruit peels of WT (left panel) or IL1B (right panel) plants.
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