Subcellular localization of the LaTPS7, LaTPS8, and LaCYP71D582 via transient expression in tobacco
When La TPS7 and La TPS8 were predicted by Signal P, the signal peptide was not detected in the plastid. However, the signal was detected when we used Phyre2, and La CYP71D582 was predicted to localize on the membranes by both Signal P and Phyre2. Hence, experimental verification is particularly important when prediction of TPSs subcellular localization is conflicted. Thus, full-ORF fusion vectors enhanced with green fluorescent protein (eGFP) were constructed for each candidate gene, and injected into N. benthaiana leaves via A. tumefaciens -mediated transfection. Analysis of whole leaves via confocal microscopy showed that both La TPS7 andLa TPS8 were associated with chloroplasts (Figure 3), whereasLa CYP71D582 was located in the endoplasmic reticulum (ER) (Figure 3).
Isolation andin-vitro enzymatic characteristic of TPSs
To determine the function of each protein, six His-tagged TPS recombinant proteins, with truncated putative signal peptides in their amino acid sequences, were expressed and extracted from E. coli BL21 (DE3). These recombinant proteins were then tested with either GPP, NPP, or FPP (10 μg for each) used as substrate. Our results show that La TPS7 catalysed GPP to produce seven monoterpenes corresponding to α -pinene, camphene, myrcene, limonene, terpinolene, linalool, and terpineol, and catalysed NPP toα -pinene, camphene, limonene, terpinolene, terpineol, and nerolidol. La TPS8 produced α -pinene, β -pinene, sylvestrene, linalool, fenchol, and geraniol from GPP, andα -pinene, limonene, terpinolene, terpineol, and nerolidol from NPP. However, no products were detected when FPP was used as substrate for both genes (Figure 4). These results indicate that these enzymes differ in their substrate recognition and product output.