Effects of overexpression of endogenous phenylalanine ammonia-lyase (PALrs1) on accumulation of salidroside in Rhodiola sachalinensis

Salidroside, a novel effective adaptogenic drug extracted from the medicinal plant Rhodiola sachalinensis A. Bor, can be derived from phenylalanine or tyrosine. Due to the scarcity of R. sachalinensis and its low yield of salidroside, there is great interest in enhancing production of salidroside by the plant. In this study, a cDNA clone encoding phenylalanine ammonia-lyase (PAL) was isolated from R. sachalinensis using rapid amplification of cDNA ends. The resulting cDNA was designated PALrs1. It is 2407-bp long and encodes 710 deduced amino acid residues. Southern blot analysis of genomic DNA indicated that the PAL gene family is composed of three to five genes in the R. sachalinensis genome. Northern blot analysis revealed that transcripts of PALrs1 were present in calli, leaves and stems, but expression in roots was very low. The PALrs1 under the 35S promoter with double-enhancer sequences from CaMV-Omega and TMV-Omega fragments was transferred into R. sachalinensis via Agrobacterium tumefaciens. PCR and PCR-Southern blot confirmed that the PALrs1 gene had been integrated into the genome of transgenic plants. Northern blot analysis revealed that the PALrs1 gene had been expressed at the transcriptional level. High-performance liquid chromatography indicated that overexpression of the PALrs1 gene resulted in a 3.3-fold increase in p-coumaric acid content, as expected. In contrast, levels of tyrosol and salidroside were 4.7-fold and 7.7-fold, respectively, lower in PALrs1 transgenic plants than in controls. Furthermore, overexpression of the PALrs1 gene resulted in a 2.6-fold decrease in tyrosine content. These data suggest that overexpression of the PALrs1 gene and accumulation of p-coumaric acid did not facilitate tyrosol biosynthesis; tyrosol, as a phenylethanoid derivative, is not derived from phenylalanine; and reduced availability of tyrosine most likely resulted in a large reduction in tyrosol biosynthesis and accumulation of salidroside.