Preliminary Study On The Molecular Mechanism Of Fruit Coloration In The Red Bud Sport Of 'Ralls' Apple

Apple (Malus×domestica Borkh) is one of several main types of fresh fruits in the world. Skin color is an important component of fruit quality and significantly influences the market value of apple fruits.'Ralls'apple is one of the apple cultivars that widely planted in northern China in 1980s. It has the characters of late maturing, good flavors and strong stress resistance and has regained popularity with people in recent years. But poor coloration is the main limiting factor of its development. In 2005, we found a mutation which had been proved to be a bud sport of'Ralls'. Fruits of the bud sport colored eailier and better, and were blushed-red at ripening. The degree of red coloration in apple skin is determined by the content and composition of anthocyanins, which are secondary metabolites synthesized through a series of enzymatic reactions. Previous work had studied the accumulation of anthocyanins and the activity of key enzymes in the biosynthetic pathway compared with'Ralls'apple. In this study, we compared the transcript levels of anthocyanin biosynthetic and regulatory genes during late developing stage, cloned and compared the coding regions of MdMYB1, MdbHLH3 and MdbHLH33 to find out the key gene that caused the mutation. We also compared the genomic sequences and upstream, downstream regions of MdMYB1 and methylation status of MdMYB1 promoter region to explain the reason for the increasing of MdMYB1expression in the bud sport. The results are shown as follows:1. Under normal sunlight condition, the transcript levels of MdCHS, MdF3H and MdUFGT in the skin tissues of fruits increased gradually during late developmental stage till harvesting, but that of MdCHI, MdDFR1 and MdLDOX peaked earlier and then declined to a lower level. The transcript levels of six anthocyanin biosynthetic genes in the fruits covered in bags were very low, but induced greatly after bag removal. Compared with those under normal sunlight, the transcript levels of MdDFR1, MdLDOX and MdUFGT were significantly improved in bagging treated fruits; but MdCHS, MdCHI and MdF3H showed little differece. Take a comparison between the two varieties, transcript levels of the biosynthetic genes in the bud sport were significantly higher than that in'Ralls'; the differences were more obvious in late genes of the pathway. In the other two branches of flavonoid biosynthesis, the transcript levels of MdFLS in the bud sport were significantly lower than that in'Ralls'. The transcription of MdLAR1and MdANR showed little difference between the two materials. They were a little lower in the bud sport only at one stage.2. Specific primers were designed according to the cDNA regions of MdMYB1, MdbHLH3 and MdbHLH33. The coding regions of the three common regulatory genes were isolated from both'Ralls'and its bud sport. Sequence comparison revealed that the coding regions of MdMYB1 and MdbHLH3 were identical between the two varieties. So the transcription factors they encode were of the same structure. The amino acid residues at the 304 position of transcription factor MdbHLH33 were different: it was Met in'Ralls'while Thr in the bud sport.3. Analysis of transcription of MdMYB1, MdbHLH3 and MdbHLH33 suggested that: under normal sunlight, the transcript level of MdMYB1 increased progressively during late developing stage, reached its maximum level before harvesting and then declined to a lower level. MdMYB1 transcript level in the fruits covered in bags was very low, but induced greatly after bag removal. Compared with those fruits under normal sunlight, the transcript level of MdMYB1 was greatly improved in those bagging treated fruits. Take a comparison between the two varieties, the transcript level of MdMYB1 in the bud sport was significantly higher than'Ralls'under both treatments. The transcription profile of MdbHLH3 did not show any link with the accumulation of anthocyanin. Whereas MdbHLH33 shared the similar transcription profile with that of MdMYB1. However, both their transcript levels showed no significant differences between the two apple varieties.4. The genomic sequences and upstream, downstream regions of MdMYB1 were isolated and compared. Analysis by agarose gel electrophoresis showed no size differences between the corresponding fragments of the two cultivars; sequence alignment also showed no significant differences between different varieties. Therefore, there were no large insertions or deletions like transposable element occurred in the DNA sequence of MdMYB1 in the bud sport. Besides,the methylation status of MdMYB1 promoter region was discovered much lower in the bud sport.5. From the findings above, it can be concluded that the transcription of all the biosynthetic genes was coordinately up-regulated; elevation of MdMYB1 transcription was the main reason that caused the up-regulation of biosynthetic genes; the lowered methylation level of MdMYB1 promoter region resulted in the elevation of MdMYB1 transcription.