Chalcone synthase is a key and often rate-limiting enzyme in the biosynthesis of anthocyanin pigments that accumulate in plant organs such as flowers and fruits, but the relationship between CHS expression and the petal coloration level in different cultivars is still unclear. In this study, three typical crabapple cultivars were chosen based on different petal colors and coloration patterns. The two extreme color cultivars, ‘Royalty’ and ‘Flame’, have dark red and white petals respectively, while the intermediate cultivar ‘Radiant’ has pink petals. We detected the flavoniods accumulation and the expression levels of McCHS during petals expansion process in different cultivars. The results showed McCHS have their special expression patterns in each tested cultivars, and is responsible for the red coloration and color variation in crabapple petals, especially for color fade process in ‘Radiant’. Furthermore, tobacco plants constitutively expressing McCHS displayed a higher anthocyanins accumulation and a deeper red petal color compared with control untransformed lines. Moreover, the expression levels of several anthocyanin biosynthetic genes were higher in the transgenic McCHS overexpressing tobacco lines than in the control plants. A close relationship was observed between the expression of McCHS and the transcription factors McMYB4 and McMYB5 during petals development in different crabapple cultivars, suggesting that the expression of McCHS was regulated by these transcription factors. We conclude that the endogenous McCHS gene is a critical factor in the regulation of anthocyanin biosynthesis during petal coloration in Malus crabapple.

Virus-induced gene silencing (VIGS) is a valuable reverse genetics tool to study gene function in a broad range of plants. As the efficiency of apple stable transformation is very low and it is still not easy to produce a large number of apple transgenic plants for most laboratories. Here, we developed an easy and effective transient transformation method of agro-inoculation for virus-induced gene silencing based on vacuum infiltration in genus of apples, the crabapple. The entire young tissue cultured crabapple plantlets were used as the plant material for infiltration, and a -90 kPa vacuum pressure and 19°C day/ 18°C night cultivation temperature were shown to result in highest VIGS efficiency. Infiltration with tobacco rattle virus containing a fragment of the transcription factor gene McMYB10, which regulates the synthesis of anthocyanin pigments, resulted in low levels of McMYB10 transcripts and characteristic faded red leaves. In addition, the expression of several anthocyanin biosynthetic genes was down-regulated in the transformed leaves. The results of this study indicate that the MYB10 gene represents a useful reporter gene for VIGS in crabapple and that the VIGS technique can be successfully applied to crabapple, thereby providing a platform for functional genetic studies in this and likely other Malus species.