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Background  Labdane-related diterpenoids form the largest group among the diterpenes. They fulfill important functions in primary metabolism as essential plant growth hormones and are known to function in secondary metabolism as, for example, phytoalexins. The biosynthesis of labdane-related diterpenes is mediated by the action of class II and class I diterpene synthases. Although terpene synthases have been well investigated in poplar, little is known about diterpene formation in this woody perennial plant species.    Results  The recently sequenced genome of  Populus trichocarpa  possesses two putative copalyl diphosphate synthase genes ( CPS , class II) and two putative kaurene synthase genes ( KS , class I), which most likely arose through a genome duplication and a recent tandem gene duplication, respectively. We showed that the  CPS -like gene  PtTPS17  encodes an  ent -copalyl diphosphate synthase ( ent -CPS), while the protein encoded by the putative  CPS  gene  PtTPS18  showed no enzymatic activity. The putative kaurene synthases PtTPS19 and PtTPS20 both accepted  ent -copalyl diphosphate ( ent -CPP) as substrate. However, despite their high sequence similarity, they produced different diterpene products. While PtTPS19 formed exclusively  ent -kaurene, PtTPS20 generated mainly the diterpene alcohol, 16α-hydroxy- ent- kaurane. Using homology-based structure modeling and site-directed mutagenesis, we demonstrated that one amino acid residue determines the different product specificity of PtTPS19 and PtTPS20. A reciprocal exchange of methionine 607 and threonine 607 in the active sites of PtTPS19 and PtTPS20, respectively, led to a complete interconversion of the enzyme product profiles. Gene expression analysis revealed that the diterpene synthase genes characterized showed organ-specific expression with the highest abundance of  PtTPS17  and  PtTPS20  transcripts in poplar roots.    Conclusions  The poplar diterpene synthases PtTPS17, PtTPS19, and PtTPS20 contribute to the production of  ent -kaurene and 16α-hydroxy- ent- kaurane in poplar. While  ent -kaurene most likely serves as the universal precursor for gibberellins, the function of 16α-hydroxy- ent- kaurane in poplar is not known yet. However, the high expression levels of  PtTPS20  and  PtTPS17  in poplar roots may indicate an important function of 16α-hydroxy- ent- kaurane in secondary metabolism in this plant organ.    Electronic supplementary material  The online version of this article (doi:10.1186/s12870-015-0647-6) contains supplementary material, which is available to authorized users.

One amino acid makes the difference: the formation of ent-kaurene and 16α-hydroxy-ent-kaurane by diterpene synthases in poplar