Chauncey R. Benedict

The enzymatic formation of δ-cadinene from farnesyl diphosphate in extracts of cotton

Abstract Incubation of extracts of cotton stele tissue which had been infected with Verticillium dahliae led to the enzymatic incorporation of [1-3H]-farnesyl diphosphate into hydrocarbons soluble in hexane-ethyl acetate. The mixture of [3H]-hydrocarbons was separated into components by HPLC. The hydrocarbon with a retention time of 17.5–18.0 min, biosynthetically prepared from (1RS)-[1- 2 H ]-(E,E)- farnesyl diphosphate, was analysed by GC-MS. The data not only agree with the reported mass spectrum of δ-cadinene but also support the proposed hydride shift in the biosynthesis of δ-cadinene from farnesyl diphosphate.

The enzymatic cyclization of nerolidyl diphosphate by δ-cadinene synthase from cotton stele tissue infected with verticillium dahliae

Abstract Soluble preparations of cotton stele tissue infected with Verticillium dahliae containing δ-cadinene synthase convert (1- RS )-[1- 2 H]- E,E -farnesyl diphosphate to [5- 2 H]- and [11- 2 H]-δ-cadinene and convert [4,4,13,13,13- 2 H 5 ]-nerolidyl diphosphate to [8,8,15,15,15- 2 H 5 ]-δ-cadinene. These data imply that nerolidyl diphosphate is an intermediate in the enzymatic cyclization of the natural substrate E,E -farnesyl diphosphate to δ-cadinene by δ-cadinene synthase and involves the conversion of E,E -farnesyl diphosphate to nerolidyl diphosphate followed by cyclization to cis -germacradienyl cation, a 1,3-hydride shift, a second cyclization to a cadinanyl cation and deprotonation to δ-cadinene. Kinetic analyses of induced δ-cadinene synthase mRNA, δ-cadinene synthase activity and formation of sesquiterpenoid phytoalexins in cotton stele tissue infected with Verticillium dahliae show that 12 hr after fungal inoculation the δ-cadinene synthase mRNA was at a maximum level. The tissue injected with H 2 O in place of fungal inoculation showed no detectable δ-cadinene synthase mRNA or δ-cadinene synthase activity after 12 to 96 hr. After 12 hr, 54% of the δ-cadinene synthase activity had developed, but no phytoalexins were detected, the midpoint in the formation of the phytoalexins was 48 hr. These data, together with the enzyme analyses, support the conclusion that Verticillium dahliae initiates a signal in the stele tissue that results in an increased steady-state level of δ-cadinene synthase mRNA and an increased activity of δ-cadinene synthase which functions in the conversion of E,E -farnesyl diphosphate → nerolidyl diphosphate → δ-cadinene that is metabolically converted to desoxyhemigossypol, desoxyhemigossypol-6-methyl ether, hemigossypol and hemigossypol-6-methyl ether.

Reduced levels of cadinane sesquiterpenoids in cotton plants expressing antisense (+)-δ-cadinene synthase

Cotton plants were transformed with an antisense construct of cdn1-Cl, a member of a complex gene family of delta-(+)cadinene (CDN) synthase. This synthase catalyzes the cyclization of (E,E)-farnesyl diphosphate to form CDN, and in cotton, it occupies the committed step in the biosynthesis of cadinane sesquiterpenoids and heliocides (sesterterpenoids). Southern analyses of the digestion of leaf DNA from R(o), T(o), and T(1) plants with Hind III, Pst I and Kpn I restriction enzymes show the integration of antisense cdn1-C1 cDNA driven by the CaMV 35S promoter into the cotton genome. Northern blots demonstrate the appearance of cdn synthase mRNA preceding CDN synthase activity and the formation of gossypol in developing cottonseed. T(2) cottonseed show a reduced CDN synthase activity and up to a 70% reduction in gossypol. In T(1) leaves the accumulated amounts of gossypol, hemigossypolone and heliocides are reduced 92.4, 83.3 and 68.4%, respectively. These data demonstrate that the integration of antisense cdn1-C1 cDNA into the cotton genome leads to a reduction of CDN synthase activity and negatively impacts on the biosynthesis of cadinane sesquiterpenoids and heliocides in cotton plants.

Synthesis of a tritium labeled photolabile analogue of farnesyl diphosphate: (E, E)-[1-3H]-(2-diazo-3-trifluoropropionyloxy)geranyl diphosphate (DATFP-GDP)

Tritiated (E, E)-(2-diazo-3-trifluoropropionyloxy)geranyl diphosphate (DATFP-GDP) has been used as a photolabile analogue of (E, E)-farnesyl diphosphate (E, E-FDP) for an aid in isolating enzymes utilizing E, E-FDP as a substrate. We now report an alternative method of synthesizing this probe in which the tritium label is introduced in the step just before the introduction of the diphosphate group. Thus, DATFP-geraniol is oxidized to DATFP-geranial with activated manganese dioxide. The tritium label is introduced by reduction of the aldehyde with NaBT4. The DATFP-group successfully withstands both of these steps. The overall yield for these two steps is 69%. Diphosphorylation of the resulting alcohol afforded DATFP-[1-3H]-GDP in 8% yield with a specific activity of 48.6 μCi/μmol and radiochemical purity of 94%.

The chemical regulation of polyisoprenoid synthesis in Parthenium argentatum with 2-(3,4-dichlorophenoxy)-triethylamine

Applications of 2-(3,4-dichlorophenoxy)-triethylamine (DCPTA) to guayule plants (Parthenium argentatum) increase the enzymatic polymerization of isopentenyl pyrophosphate (IPP) to polyisoprene. The purpose of this study was to determine the effectiveness of DCPTA in increasing polyisoprenoid rubber synthesis at the end of the winter period of rubber synthesis in plants expressing a high level of polymerization activity. Guayule seedlings were transplanted to desert field plots in May, 1987 and treated with DCPTA. The polymerization activity in the stems was monitored throughout the year and the rubber content of the stems was determined in April, 1988. Treatment of plants with DCPTA increased the enzymatic polymerization activity in stem homogenates. The polymerization activity in control plants increased from 4 nmol h−1 g fresh wt.−1 in July to 183 nmol h−1 g fresh wt.−1 in January and the activity in plants with 1000 ppm DCPTA increased from 7 nmol h−1 g fresh wt−1 in July to 340 nmol h−1 g fresh wt.−1 in January. The high mol.wt. rubber polymer in the stems was determined by solvent extraction and 13C-NMR spectroscopy. Applications of different concentrations of DCPTA to guayule plants resulted in a statistically significant increase of 7–33% of the rubber content of the stems. The application of DCPTA to guayule seedlings stimulates the synthetic pathway for the production of secondary products in mature plants.