Expression and promoter activity analysis of PpCCD4 closely related to carotenoid synthesis in peach

Abstract:【Objective】Carotenoids are important terpenoids in the secondary metabolites of plants. They not only have the function of protecting the plant itself by photoprotection and removal of reactive oxygen species, but also are important precursors of vitamins synthesized by the human body. Based on the color change of the yellow-flesh and the -flesh peach (Amygdalus persica L.) fruits during ripening, the correlation between the total carotenoids and the expression of the gene PpCCD4 was analyzed to explore the difference of the PpCCD4 promoter activity among different varieties, and then to explain the expression differences of the PpCCD4 in the yellow-flesh fruits.【Methods】The representative vari- eties of yellow flesh peach‘Golden Baby 6’and white flesh peach‘Yan Hong’were selected. The total carotenoid contents were measured at significant periods of fruit phenotypic change of the two cultivars, and specific primers were designed to analyze the fruit developmental period. Correlation analysis between the changes of total carotenoid contents and the expressions of the PpCCD4 in the flesh was made, specific primers were designed according to the peach reference genome. The differences of thePpCCD4 transcripts of the different peach varieties were detected. The sequences of PpCCD4 and pro-moter were cloned. The analysis of sequences and analysis of promoter regulatory elements was subse- quently made. Using homologous recombination technology, the separately recovered promoter se- quences were ligated with the digested pCAMBIA1301 vector plasmid to construct a PpCCD4 promot- er to drive the GUS expression vector and to transform Agrobacterium to infect the peach pulp. The two promoters were analyzed according to the GUS staining of the pulp. The different activity of the promot- ers was employed to explain the difference of the expression of PpCCD4 during the fruit development.【Results】The total amount of carotenoids in the white flesh fruits during maturity was reduced to 6.6μg · g- 1, and the total amount of carotenoids in the yellow flesh fruits was increased to 334.46 μg · g- 1. During the period of 40 d to 85 d after flowering, the expression of the PpCCD4 in the fruits of‘Yan- hong’was significantly up-regulated. The fruit development approached to full maturity on 85 d, after flowering, the expression of PpCCD4 approached zero, and the expression of PpCCD4 in‘Golden Ba- by 6’fruits was low and hardly changed. There was a significant difference in the expression ofPpCCD4 between the yellow flesh and the bwhite flesh. On At 65 d after flowering, the peak expres- sion of the PpCCD4 in the fruits of‘Yanhong’reached 13.6 times more than that of‘Golden Baby 6’. There was a correlation between the expression of PpCCD4 and carotenoids during the development of‘Yanhong’(r2> 0.8). The total carotenoid content of‘Golden Baby 6’had no correlation with the expression of PpCCD4. In the yellow flesh variety, the PpCCD4 had two base insertions in the CDS region compared with the white flesh variety.‘Golden Baby 6’inserted a TC repeat at position 864 bp from the gene’s starting point, and the TC insertion in this transcription mode caused a frameshift. The mutation caused premature termination of transcription, resulting in the loss of the function of the en- zyme that degraded carotenoids encoded by the PpCCD4, and inducing accumulation of carotenoids in the pulp. The sequence of PpCCD4 in the white flesh peach variety‘Yanhong’did not mutate, so the sequence of the amino acid was not altered and the function of the protein was normal. In addition, thePpCCD4 had alternative splicing in the yellow and the white flesh fruits. According to promoter clon- ing analysis, it was found that the promoter corresponding to the white flesh had more transcription en- hancements and promoter elements, while the yellow flesh had more stress response elements. Mean- while, GUS staining results showed that there was a significant difference in the color of GUS staining driven by the two promoters in the peach pulp, and there was a significant difference in GUS protein ac- tivity.【Conclusion】The generation of frame shift mutations in the yellow-flesh cultivars caused by the loss of the PpCCD4 function. And the flesh of the two cultivars had different PpCCD4 transcription pat- terns. Difference of promoter activity was the main reason for the significant differences of the PpCCD4 expression in the yellow-flesh and the white-flesh fruits.