We reported the functional characterization of cDNAs encoding short-chain isoprenyl diphosphate synthases that control the partitioning of precursors for lavender terpenoids.
Lavender essential oil is composed of regular and irregular monoterpenes, which are derived from linear precursors geranyl diphosphate (GPP) and lavandulyl diphosphate (LPP), respectively. Although this plant strongly expresses genes responsible for the biosynthesis of both monoterpene classes, it is unclear why regular monoterpenes dominate the oil. Here, we cloned and characterized Lavandula x intermedia cDNAs encoding geranyl diphosphate synthase (LiGPPS), geranylgeranyl diphosphate synthase (LiGGPPS) and farnesyl diphosphate synthase (LiFPPS). LiGPPS was heteromeric protein, consisting of a large subunit (LiGPPS.LSU) and a small subunit for which two different cDNAs (LiGPPS.SSU1 and LiGPPS.SSU2) were detected. Neither recombinant LiGPPS subunits was active by itself. However, when co-expressed in E. coli LiGPPS.LSU and LiGPPS.SSU1 formed an active heteromeric GPPS, while LiGPPS.LSU and LiGPPS.SSU2 did not form an active protein. Recombinant LiGGPPS, LiFPPS and LPP synthase (LPPS) proteins were active individually. Further, LiGPPS.SSU1 modified the activity of LiGGPPS (to produce GPP) in bacterial cells co-expressing both proteins. Given this, and previous evidence indicating that GPPS.SSU can modify the activity of GGPPS to GPPS in vitro and in plants, we hypothesized that LiGPPS.SSU1 modifies the activity of L. x intermedia LPP synthase (LiLPPS), thus accounting for the relatively low abundance of LPP-derived irregular monoterpenes in this plant. However, LiGPPS.SSU1 did not affect the activity of LiLPPS. These results, coupled to the observation that LiLPPS transcripts are more abundant than those of GPPS subunits in L. x intermedia flowers, suggest that regulatory mechanisms other than transcriptional control of LPPS regulate precursor partitioning in lavender flowers.
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This work was supported through grants and/or in-kind contributions to SSM by UBC, and Natural Sciences and Engineering Research Council of Canada. We thank Dr. I. Hwang, Pohang University of Science and Technology, Korea for kindly providing p326::sGFP construct DNA; Dr. Julien Gibon (Assistant Prof. at UBC Okanagan) and Dr. Phillip Barker's group for assisting with microscopy.
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Supplementary file1 (DOCX 15 kb) Fig. S1. Multiple sequence alignment of GPPS.SSU1 from angiosperms. Comparison of the predicted amino acids of LiGPPS.SSU1 and other functionally characterized plants GPPS.SSU1 subfamily. All of these GPPS.SSU1 genes are inactive by themselves. They contain the two conserved 'CxxxC' motifs, essential for the physical interaction of different subunits, and lack the two conserved aspartate-rich motifs that function in catalytic and substrate binding. L. x intermedia GPPS.SSU1 shares the highest amino acids identity with M. x piperita GPPS.SSU1 (67.35%) and the lowest with C. breweri GPPS.SSU1 (43.2%) and H. lupulus GPPS.SSU1 (44%).
Supplementary file2 (DOCX 14 kb) Fig. S2. The predicted amino acid sequences alignment of LiGPPS.SSU2 and other functionally studied GPPS.SSU2 subfamily genes in plants. L. x intermedia GPPS.SSU2 shares ~70% amino acid identity with GPPS.SSU2 from Arabidopsis, and 55 % from O. sativa. Unlike these genes, L. x intermedia GPPS.SSU2 functions neither in chlorophyll synthesis nor GPP formation.
Supplementary file3 (DOCX 17 kb) Fig. S3. Alignment of amino acid sequences of LiGPPS.LSU, LiGGPPS and other functionally characterized GPPS.LSUs and GGPPSs from other selected plants. Of the given proteins, GPPS.LSU from H. lupulus and C. roseus are bifunctional as GPPS and GGPPS. Despite the presence of two aspartate-rich motifs in the sequence, M. x piperita GPPS.LSU is catalytically inactive by itself. L. x intermedia GPPS.LSU shares ~72% amino acid identity with GGPPS in the same species, and from 62 -65% identity with the rest of these genes. L. x intermedia GPPS.LSU functions as GPPS when it physically interacts with GPPS.SSU1.
Supplementary file4 (DOCX 14 kb) Fig. S4. Multiple amino acid sequence alignments of FPPSs from L. x intermedia and other selected plants. LiFPPS has the two aspartate-rich conserved motifs, but lacks any of cysteine-rich motif(s). It exhibits 69%, 78% and 92% amino acid sequence similarity to FPPS from Z. mays (ZmFPPS), A. thaliana (AtFPPS) and S. miltiorrhiza (SmFPPS), respectively.
Supplementary file5 (DOCX 359 kb) Fig. S5. Conserved motifs in L. x intermedia GPPS subunits, GGPPS and FPPS. a) GPPS/GGPPS and FPPS with transit peptide and the key conserved motif residues, b) WebLogos showing the relative frequency of the conserved motif residues found in each subfamily of short-chain trans-IDSs. The first and second aspartate-rich motifs are designated as FARM and SARM.
Supplementary file6 (DOCX 82 kb) Fig. S6. GC-MS analysis of products obtained from assays of recombinant proteins expressed in E. coli. Reaction products were (enzymatically) hydrolyzed using calf intestine alkaline phosphatase (CIP), and the resulting prenyl alcohol (geraniol) was detected by GC-MS. The left panel shows the chromatography of produces for LiGPPS.SSU1, LiGPPS.SSU2, LiGPPS.LSU and co-expressed GPPS subunits (LiGPPS.LSU/LiGPPS.SSU2 and LiGGPPS /LiGPPS.SSU2 recombinant proteins assayed with 40 µM IPP and 40 µM DMAPP, and authentic geraniol standard. The right panel indicates the mass spectrometry of geraniol standard.
Supplementary file7 (DOCX 126 kb) Fig. S7. Phylogenetic tree of amino acid sequences of the short-chain IDSs mined from N. benthamiana transcriptomic database (https://sefapps02.qut.edu.au/blast/blast_link2.cgi) and other selected plants. Scale bar indicates the numbers of substitutions per site. The GenBank accession numbers of these genes used here are listed in Table S1.
Supplementary file9 (DOCX 243 kb) Fig. S9. Dwarf phenotype complementation assay using exogenous GA3 in transgenic N. benthamiana expressing LiGPPS.SSU1. a) Phenotypes of transgenic lines compared with control (GUS) plants grown on MS media supplemented with 0 or 30 µM GA3 for two weeks. b) Plant height of the transgenic lines with and without exogenous GA3 in relative to the control plants. Exogenous GA3 improved the height of LiGPPS.SSU1 transgenic plants. ** P< 0.01, Data are means ± standard error (n=6 plants per transgenic line). ns = non-significant.
Supplementary file12 (DOCX 12 kb) Table S3. Plant phenotypes of transgenic lines expressing LiGPPS.SSU2. All LiGPPS.SSU2 transformed lines displayed similar growth phenotypes to the control plants. Data are means ± standard error (n = 3), ns - non-significant at p< 0.05, Student's t-test. All plant growth parameters were collected at 8 weeks after seeding
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Adal, A.M., Mahmoud, S.S. Short-chain isoprenyl diphosphate synthases of lavender (Lavandula). Plant Mol Biol 102, 517–535 (2020). https://doi.org/10.1007/s11103-020-00962-8
- Geranyl diphosphate synthase
- Precursor partitioning
- Regular monoterpenes