Functional Characterization Of Tyrosinase Gene BvcTYR From Red Swiss Chard

Betalains are water-soluble nitrogen-containing pigments that color plants as anthocyanins but exclusively in certain families of one single order, Caryophyllales. They are widely used in food industry as colorant and in pharmaceutical industry as antioxidant and radical scavenging agents against certain oxidative stress-related disorders. They are believed derived from tyrosine through three main enzyme-mediated steps. The first and even the second steps, L-tyrosine hydroxylation and L-DOPA oxidation, were proposed to be mediated by tyrosinase, but the molecular evidence remains lacking. A tyrosinase gene, BvcTYR, from red Swiss chard was previously isolatedBvcTYR encodes a PPO-type tyrosinase that may is cytoplasm-localized. In red Swiss chard, BvcTYR enzymatic activities toward L-tyrosine and L-DOPA are higher in the root than in petiole and leaf-blade, which is consistent with BvcTYR transcript abundance and betalain contents. The transcript abundance varied with the developmental stages, and the highest one appeared at 8-leaf stage in the root, but at 10-leaf stage in both petiole and leaf-blade. For the enzymatic activities and betalain content, the peak emerged at 10-leaf stage, one stage later than the transcript abundance. Thus a good correlation is present among the BvcTYR transcript abundance, BvcTYR enzymatic activities and betalain content.Ectopic over-expression of BvcTYR in anthocyanin-producing tobacco conferred transgenic plants the capability to hydroxylate L-tyrosine and increased their enzymatic activity to L-DOPA, and RNAi silence of the transgene reduced significantly BvcTYR transcript abundance and decraesed its enzymatic activity.Transient over-expression of BvcTYR in green chard augmented BvcTYR transcript abundance by 2 times, and increased the monophenolase activity and diphenolase one by 20% and 95%, respectively, although didn’t change plant color. RNAi-silencing of BvcTYR in the red petiole of red chard turned the red color to yellowish. In green leaf of Red-Petiole, the infection with empty vector (EV) and RNAi-BvcTYR stimulated betacyanins synthesis, increased the betalain content, BvcTYR transcript abundance and both monophenolase activity to toward L-tyrosine and diphenolase activity to L-DOPA, but the growth of RNAi is less than EV because of inhibited the activities of BvcTYR. RNAi knock-down of BvcTYR in red-leaf of red chard resulted in loss of red color.Together, these data suggest that the tyrosinase encoded by BvcTYR mediates may at least the hydroxylation of L-tyrosine the first step of the biosynthetic pathway of betalains. Thus, this discovery provided the first molecular evidence to tyrosinase’s involvement in the betalain biosynthesis and will make it possible to engineer the pathway into heterologous species and in particular into non-betalain species.