Part 3 Combining the information to find the causal gene for pink fruit colour

From the first part of the assignment you learned that the gene that determines red or pink fruit colour is located on chromosome 1, between 44.5 and 72.4 Mb on chromosome 1 of tomato. From part 2 you learned that the red fruits, in addition to lycopene, also accumulate a yellow flavonoid, whereas this flavonoid is absent in pink fruits of IL1B.

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.

The expression of structural genes encoding enzymes of a metabolic pathway is regulated by so-called transcription factor genes. The flavonoid pathway genes are regulated by a complex of three transcription factor (TF) genes: MYB, basic Helix-Loop-Helix (bHLH) and WD40 repeat (WDR) transcription factor genes. The encoded proteins form a complex that activates transcription of the structural gene promoters (Figure 5A). MYB and bHLH TF’s interact with each other and bind to sequence elements in the promoters of the structural genes. They determine the specificity, i.e. which genes are activated and which are not. The WDR protein stabilises the complex. For regulation of the pathway leading to flavonols, which is active in tomato fruit peel, it has been shown that a MYB transcription factor alone is sufficient to activate the target genes (Figure 5B). 

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Figure 5. Models for the participation of MYB, bHLH and WDR trancription factors in the regulation of the flavonoid pathways. A. Regulation of the pathway to anthocyanins; B. Regulation of the pathway to flavonols.

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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• Is any of the tested genes located in the CH1B region?

• If you look at the pathway depicted in Figure 4, which part of the pathway did we NOT check?

Assuming that there are no structural pathway genes located in the IL1B region, it is possible that this region contains a transcription factor gene that regulates the expression of the structural genes in the flavonoid pathway. In that case we should see a difference in expression of one or more flavonoid genes in WT fruits compared to IL1B fruits. In worksheet Flavonoid gene expression you see the results of a microarray experiment in which the mRNA expression in WT fruit peel was compared with that of IL1B fruit peel. Expression was monitored in four different ripening stages: Green (G), Breaker (B), Turning (T) and Ripe (R) fruits. Figure 6 shows the expression of these genes in WT and IL1B fruit peel.

• 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.

• As mentioned above it is known that MYB transcription factors regulate the flavonoid pathway. How many MYB transcription factor genes are present in the IL1B introgression region? Hint: Use the filter function in Excel. Filter in column C the genes between 44 and 72Mb, followed by a filtering of column D with the keyword MYB.

We hypothesise that one of these transcription factor genes is involved in the regulation of the flavonoid pathway in tomato fruit. We can expect that the expression pattern of the structural flavonoid genes correlates well with that of the transcription factor gene that is regulating them. In the worksheet MYB gene expression you see the expression patterns of the four transcription factor genes in the IL1B region in ripening fruit peels of WT and IL1B fruits.

• Which of these four MYB genes are well expressed in fruit peels?

• Which of these genes would be your favourite candidate gene and why?

• Calculate the correlation coefficient of the expression of Solyc01g079620 and CHS2 using the Correl function in Excel.

• What is the annotation of Solyc01g079620? Check the function of this gene in Google. What does it regulate?

The combined analysis of genetic data, biochemical data, biochemical pathway, gene position and gene expression data led to discovery of the MYB12 gene as causal gene for the pink mutation. The results clearly show that the MYB12 gene in IL1B is not expressed upon ripening, resulting in a low expression of the flavonoid pathway and a lack of accumulation of the yellow-coloured naringenin chalcone. These results, however, are all based on association of the gene position, gene annotation and gene expression with the yellow pigmentation. But this is not a real proof. Which experiment would you design to further proof that this gene is indeed the causal gene of the pink mutation?