The cis-prenyltransferase protein family in Taraxacum koksaghyz

Boje Müller

        “As the first comprehensive analysis of the cis-prenyltransferase family in T. koksaghyz, our study provides unparalleled insights into the regulation of isoprenoid metabolism in this natural rubber-producing species. Our study emphasizes the rubber transferase complex's specialization and paves the way for the development of improved, targeted enzymes."

With tens of thousands of compounds, isoprenoids constitute one of the largest groups of plant secondary metabolites. Their structure is formed by the sequential addition of the C5 isoprene unit isopentenyl diphosphate (IPP). Isoprenoid synthesis is initiated by the condensation of IPP with its isomer dimethylallyl phosphate (DMAPP). Longer-chain linear polyisoprenoids are formed through successive head-to-tail additions of IPP. A class of enzymes known as prenyltransferases catalyze the sequential transfer of IPP units to allylic diphosphate acceptors. Depending on the stereochemistry of the polyisoprenoid product, these enzymes are classified either as trans-prenyltransferases (transPTs) or as cis-prenyltransferases (cisPTs).

The researchers characterized the family of eight cis-prenyltransferases in Taraxacum koksaghyz (TkCPT1–8) to better understand their expression, subcellular localization, interactions with TkCPTL proteins (cis-prenyltransferase-like, CPTL), and physiological functions.

Using qRT-PCR, mRNA expression profiles for TkCPT1–8 were generated, showing that TkCPT1 and TkCPT2 mRNAs were most abundant in latex, whereas TkCPT3 and TkCPT6–8 mRNAs predominantly appeared in leaves. TkCPT3 was expressed at higher levels than TkCPT6–8. TkCPT4 was constitutively expressed at low levels in all tissues. In contrast, TkCPT5 mRNA was only detected at high concentrations in flowers. To investigate subcellular localization, researchers employed transient co-expression with specific fluorescently labeled proteins in Nicotiana benthamiana epidermal cells. Confocal laser scanning microscopy analysis revealed that TkCPT1–4 are localized in the endoplasmic reticulum and TkCPT5–7 are localized in the chloroplasts.

The interaction analyses with TkCPT1–7 proteins were also performed in Nicotiana benthamiana using bimolecular fluorescence complementation. A fluorophore (mRFP) is split into two non-fluorescent halves, which are used to label the potential interaction partners. If the proteins interact with each other, the two halves of the fluorophore (mRFP) bind to each other and fluoresce red when excited by green light. Co-expressing TkCPT3 or TkCPT4, each fused to one half of mRFP, with TkCPTL1 or TkCPTL2, fused to the other half, resulted in strong red fluorescence.

This suggests that TkCPT3 and TkCPT4 both interact with TkCPTL1/TkCPTL2. The researchers demonstrated that TkCPT5–8 does not interact with TkCPTL1/TkCPTL2. The production of various linear isoprenoids was detected for all enzymes in the recombinant yeast system. TkCPT1 and TkCPT2 together with TkCPTL1form polyisoprenoids; TkCPT3 and TkCPT4 (with TkCPTL1) generate shorter-chain polyisoprenoids that are converted into dolicholes by other enzymes. TkCPT5 produces pentaprenol, TkCPT6 nonaprenol, and TkCPT7 and TkCPT8 nerol.