Monoterpenes in the glandular trichomes of tomato are synthesized from a neryl diphosphate precursor rather than geranyl diphosphate

Abstract

We identified a cis-prenyltransferase gene, neryl diphosphate synthase 1 (NDPS1), that is expressed in cultivated tomato (Solanum lycopersicum) cultivar M82 type VI glandular trichomes and encodes an enzyme that catalyzes the formation of neryl diphosphate from isopentenyl diphosphate and dimethylallyl diphosphate. mRNA for a terpene synthase gene, phellandrene synthase 1 (PHS1), was also identified in these glands. It encodes an enzyme that uses neryl diphosphate to produce beta-phellandrene as the major product as well as a variety of other monoterpenes. The profile of monoterpenes produced by PHS1 is identical with the monoterpenes found in type VI glands. PHS1 and NDPS1 map to chromosome 8, and the presence of a segment of chromosome 8 derived from Solanum pennellii LA0716 causes conversion from the M82 gland monoterpene pattern to that characteristic of LA0716 plants. The data indicate that, contrary to the textbook view of geranyl diphosphate as the "universal" substrate of monoterpene synthases, in tomato glands neryl diphosphate serves as a precursor for the synthesis of monoterpenes.

Fig. 1.

Monoterpenes obtained from S. lycopersicum (M82) and S. pennellii (LA0716) plants and two isogenic chromosomal substitution lines and analyzed by GC-MS. (A) A leaf was dipped briefly in MTBE and the extract analyzed. (B) Type VI trichomes were collected by hand and placed into a vial containing MTBE. Numbered peaks are as follows: 1, δ-2-carene; 2, α-phellandrene; 3, α-terpinene; 4, limonene; 5, β-phellandrene; 6, γ-terpinene. No linalool was detected in any of the samples. Chromatograms show the detection of m/z = 93. Rt, retention time.

Fig. 2.

Quantitative RT-PCR analysis of NDPS1 and PHS1 gene expression in M82 and LA0716 trichomes. Expression of NDPS1 and PHS1 was measured by qRT-PCR and normalized to expression of EF-1α in total trichomes isolated from stem and petiole tissue and from stem and petiole tissue after removal of the trichomes.

Fig. 3.

In vitro assay for identification of NDPS1 product using GC-MS. (A) Purified NDPS1 was incubated with IPP and DMAPP for 30 min at 30 °C, after which alkaline phosphatase was added to the reaction and the solution incubated overnight and then analyzed. (B) Authentic nerol standard. (C) Authentic geraniol standard. (D) Commercial GPP was treated with alkaline phosphatase as in A. GC-MS chromatograms show the detection of m/z = 93.

Fig. 4.

In vitro assays with purified recombinant PHS1 using different substrates. (A) S. lycopersicum (M82) monoterpene profile obtained by dipping a leaf in MTBE. Peaks labeled 1–5 are the same as in Fig. 1A. (B) GC analysis of the in vitro-coupled reaction catalyzed by NDPS1 and PHS1 and using IPP and DMAPP as substrates. (C) GC analysis of products of the reaction catalyzed by PHS1 with NPP as the substrate. (D) GC analysis of products of the reaction catalyzed by PHS1 with GPP as the substrate. Labeled peaks are as follows: 7, myrcene; 8 and 9, ocimene isomers. A small linalool peak (equivalent to the size of α-phellandrene peak (peak 3) in this chromatograph is also observed at retention time (Rt) 17.4 min (not shown here). GC-MS chromatograms show the detection of m/z = 93. Heights of peaks are not comparable between samples.

Fig. 5.

A model showing how GDPS and NDPS could produce their respective products from IPP and DMAPP by extracting a different proton at C4 of the IPP molecule during the condensation reaction (based on ref. 29).

Fig. 6.

Proposed mechanism for the synthesis of monoterpenes when PHS1 uses GPP or NPP as substrates. A minor peak of α-pinene is occasionally seen in the in vitro assay of PHS1 with NPP, and a correspondingly small portion of α-pinene is also occasionally seen in the profile of monoterpenes extracted from S. lycopersicum leaves.