Comparative analysis of three SmHPPR genes encoding hydroxyphenylpyruvate reductases in Salvia miltiorrhiza

Hydroxyphenylpyruvate reductase (HPPR) is an enzyme that is involved in the biosynthesis of hydrophilic phenolic acids in Salvia miltiorrhiza, which is a model medicinal plant. Three SmHPPR genes have been identified in the S. miltiorrhiza genome; however, only one has been functionally analyzed. Here, we cloned three SmHPPR genes (SmHPPR1, SmHPPR2, and SmHPPR3) from the cDNA of S. miltiorrhiza, and their expression profiles were studied. The expression levels of SmHPPR1 were significantly higher than those of SmHPPR2 and SmHPPR3, where SmHPPR1 revealed the highest level in stems, while SmHPPR2 and SmHPPR3 exhibited the highest level in flowers. SmHPPR1, SmHPPR2, and SmHPPR3 are localized in the cytoplasm. All three recombinant enzymes had HPPR activities and catalyzed the reduction of 4-hydroxyphenylpyruvic acid (pHPP) to 4-hydroxyphenyllactic acid (pHPL), with SmHPPR1 showing the highest activity. The transient over-expression of SmHPPR1, SmHPPR2, and SmHPPR3 in the leaves of Nicotiana benthamiana promoted the production of pHPL, which indicated that all three SmHPPRs had in vivo activities. Overall, between the three homologs, SmHPPR1 plays a dominant role in catalyzing pHPP to pHPL, which provides new insights into the biosynthesis of phenolic acids in S. miltiorrhiza.