Functional Analysis Of PhACC1, PhACC2 And PhAAE13 During The Anthocyanin Biosynthesis In The Petunia

Petunia hybrida is one of the most important models to study plant organ development and flower color. Anthocyanin is one of the main pigments that form flower color. Many structural genes of anthocyanin biosynthesis, such as CHS, CHI, F3 H, DFR, ANS and UFGT, have been cloned and identified. Acetyl-Co A carboxylase(ACC) catalyzes acetyl-Co A and CO2 to generate malonyl-Co A under the participation of ATP. Recent studies suggested that malonyl-Co A synthetase(Acyl Activating Enzyme, AAE) could catalyze malonic acid to generate malonyl-Co A, providing precursor for the biosynthesis of anthocyanin. So these two enzymes could be the key enzyme of anthocyanin biosynthesis. Previous studies of ACC mainly concentrated on fatty acid biosynthesis. However, its effect on anthocyanin biosynthesis has not been reported. The role of AAE in anthocyanin biosynthesis has not been reported, too. In addition, the MATE is one of the important membrane transporters. Studies have shown that thistles alfalfa MATE2 has strong anthocyanin transport capacity and the role of MATE in petunia anthocyanin transport has not been reported.This study aims to explore the function of PhACC1, PhACC2, PhAAE13, PhAAE3, PhAAE14, PhMATE1 and PhMATE2 during anthocyanin synthesis in the petunia.This study first obtained the fragments of PhACC1, PhACC2, PhAAE13, PhAAE3, PhAAE14, PhMATE1 and PhMATE2. The recombinant vectors p TRV2-PhACC1, p TRV2-PhACC2, p TRV2-PhAAE13, p TRV2-PhAAE3, p TRV2-PhAAE14, p TRV2-PhMATE1 and p TRV2-PhMATE2 were successfully constructed. Then these genes silenced petunia plants were obtained by using VIGS technique. To analyse the functional of the genes, the phenotype, including the color of petals and pistils, fruit growth, anthocyanin content and relative conductivity determination was observed. Furthermore, RNA was extracted and the quantitative real time PCR(q PCR) was used for analyzing the expression of the genes. The results are as follows:(1) PhACC1, PhACC2 and PhAAE13 silencing reduced the anthocyanin biosynthesis of petunia petals, stigmas and anthers. Petals, stigmas and anthers of control plants were purple, while those of PhACC1, PhACC2 and PhAAE13 silenced plants were pink. The level of anthocyanin reducing was not as high as PhCHS silenced plants, of which petals, stigmas and anthers were pure white. The anthocyanin content was significantly lower than the control plants. These results showed that PhACC1, PhACC2 and PhAAE13 are involved in anthocyanin biosynthesis of petunia petals, stigmas and anthers. It plays an indispensable role in the anthocyanin biosynthesis. However, silence of PhAAE3 and PhAAE14 had no significant effects on the anthocyanin biosynthesis of petunia petals, stigmas and anthers. Petals, stigmas and anthers of PhAAE3 and PhAAE14 silenced plants were purple, which were the same as control plants.(2) Silencing of PhMATE1 and PhMATE2 had no obvious influence on color of early petunia petals. In the senescence stage, petals of PhMATE1 and PhMATE2 silenced plants changed to pink under the condition of low temperature about 15? in the daytime and 10? at nigth, while petals of control plants were still purple. It showed that PhMATE1 and PhMATE2 are involved in anthocyanin transshipment of petunia petals in senescence stage.(3) Ethylene treatment significantly decreased the relative expression of PhACC1, PhACC2 and PhAAE13 in ethylene treated petals.(4) Silencing of PhACC1, PhACC2 and PhAAE13 had no obvious effects on the shape, size and thickness of petals, and the development of pistils, fruits and seeds. The shape, size and thickness of petals of PhACC1, PhACC2 and PhAAE13 silenced plants were normal. The pistils, fruits and seeds developed normally and fully. There were no significant differences compared with control plants.