Identification of a novel abscisic acid-regulated farnesol dehydrogenase from Arabidopsis

Abstract

In Arabidopsis (Arabidopsis thaliana), farnesylcysteine is oxidized to farnesal and cysteine by a membrane-associated thioether oxidase called farnesylcysteine lyase. Farnesol and farnesyl phosphate kinases have also been reported in plant membranes. Together, these observations suggest the existence of enzymes that catalyze the interconversion of farnesal and farnesol. In this report, Arabidopsis membranes are shown to possess farnesol dehydrogenase activity. In addition, a gene on chromosome 4 of the Arabidopsis genome (At4g33360), called FLDH, is shown to encode an NAD(+)-dependent dehydrogenase that oxidizes farnesol more efficiently than other prenyl alcohol substrates. FLDH expression is repressed by abscisic acid (ABA) but is increased in mutants with T-DNA insertions in the FLDH 5' flanking region. These T-DNA insertion mutants, called fldh-1 and fldh-2, are associated with an ABA-insensitive phenotype, suggesting that FLDH is a negative regulator of ABA signaling.

Figure 1.

Proposed metabolism of farnesal and farnesol as it relates to protein prenylation. The portion of the cycle shown in red is the subject of this article.

Figure 2.

Oxidation of [1-³H]farnesol to [1-³H]farnesal in the presence of Arabidopsis membranes. [1-³H]Farnesol was generated by calf intestine alkaline phosphatase digestion of [1-³H]farnesyl diphosphate and subsequently incubated with native or boiled Arabidopsis membranes in the presence of 0.1 mm NAD⁺ or NADP⁺ for 30 min at 30°C. Reaction products were resolved by silica gel TLC, visualized by fluorography (left), and compared to authentic chemical standards, which were visualized by vanillin staining (right). Retardation factor (R_f) values for farnesal (R_f = 0.59) and farnesol (R_f = 0.34) are indicated. The radioactivity at the origin is [1-³H]farnesyl diphosphate. Fal, Farnesal; Fol, farnesol.

Figure 3.

Arabidopsis farnesol dehydrogenase is specific for biologically relevant prenyl alcohol substrates. A, Farnesol dehydrogenase reactions were performed in the presence of 1 mm farnesol, geranylgeraniol, or geraniol, and reduced cofactor was detected spectrophotometrically at 340 nm as a function of time. Reduced cofactor production was linear for 2 min and was detected in the presence of farnesol or geranylgeraniol as an isoprenoid substrate. Farnesol dehydrogenase activity was determined to be 10 nmol min⁻¹ mg⁻¹. B, Farnesol dehydrogenase assays were performed as in A in the presence of 0.1% Tween 20. These data are representative of three independent experiments. The se of the mean is shown. Fol, Farnesol; GGol, geranylgeraniol; Gol, geraniol.

Figure 4.

At4g33360 (FLDH) encodes an enzyme with farnesol dehydrogenase activity. A, Membranes from SM1058 yeast cells with or without pCL194 (At5g16990), pCL195 (At5g16960), pCL196 (At4g33360), or pCL197 (At3g61220) expression constructs were assayed for farnesol dehydrogenase activity using dephosphorylated [1-³H]FPP as a substrate. B, Membranes from Arabidopsis seedlings, SM1058 cells, and SM1058/pCL196 cells were assayed for farnesol dehydrogenase activity using [1-³H]farnesol from American Radiolabeled Chemicals, which was purified by preparative TLC. Fal, Farnesal; Fol, farnesol.

Figure 5.

The FLDH-encoded farnesol dehydrogenase is NAD⁺ dependent. [1-³H]farnesol from American Radiolabeled Chemicals was used to analyze farnesol dehydrogenase activity in membranes of SM1058 and SM1058/pCL196 (At4g33360, FLDH) cells in the presence of NAD⁺ or NADP⁺ as cofactor. Fal, Farnesal.

Figure 6.

The FLDH-encoded farnesol dehydrogenase exhibits partial specificity for farnesol in competition assays. [1-³H]farnesol from American Radiolabeled Chemicals was used to analyze farnesol dehydrogenase activity in membranes from SM1058/pCL196 cells in the presence of NAD⁺ and unlabeled farnesol, geranylgeraniol, or geraniol as competitor. Ethanol was included as a solvent control. Each competitor was used at 0.33, 3.3, and 33 μm. These results are representative of two independent experiments. The se of the mean is shown. Fol, Farnesol; GGol, geranylgeraniol; Gol, geraniol.

Figure 7.

The FLDH-encoded farnesol dehydrogenase exhibits partial specificity for farnesol in spectrophotometric assays. A, Farnesol dehydrogenase reactions were performed in the presence of 1 mm farnesol, geranylgeraniol, or geraniol, and reduced cofactor was detected spectrophotometrically at 340 nm as a function of time. SM1058/pCL196 membranes were used as a source of farnesol dehydrogenase activity. B, Farnesol dehydrogenase assays were performed as in A using SM1058 membranes. These data are representative of two independent experiments. The se of the mean is shown. *, **, and *** represent significant differences compared with the SM1058 control of P < 0.05, P < 0.01, and P < 0.001, respectively, as determined by Student’s t test. Fol, Farnesol; GGol, geranylgeraniol; Gol, geraniol.

Figure 8.

FLDH is negatively regulated by ABA. A, RT-PCR was performed on 5 ng of total RNA from wild-type (Col-0) seedlings, which were grown for 4 d on 0.5× MS plates containing 1.0% Suc and 0.8% agar and then transferred to identical plates containing 0, 0.5, 2.5, or 5.0 μm cisABA for 16 h. The FLDH RT-PCR product was expected to be 693 bp in length. B, Ribosomal RNA is shown for the RNA samples used in A. Expt., Experiment.

Figure 9.

Biochemical characterization of fldh mutants of Arabidopsis. A, RT-PCR with At4g33360-RT5 and At4g33360-RT3 primers demonstrates that fldh-1and fldh-2 exhibit increased FLDH expression. B, Ethidium bromide-stained RNA used for RT-PCR in A. MW, Molecular weight. C and D, Col-0, fldh-1, and fldh-2 were analyzed spectrophotometrically for farnesol dehydrogenase activity using farnesol and NAD⁺ as substrates. The se of the mean is indicated for all data points. * and ** represent significant differences compared with the Col-0 control of P < 0.5 and P < 0.01, respectively, as determined by Student’s t test.

Figure 10.

Effects of T-DNA insertions in the 5′ flanking region of the FLDH gene on ABA signaling. A, Seed germination was scored for Col-0, fldh-1, and fldh-2 lines as a function of time in the presence of 0, 0.5, or 2.5 μm cisABA (each data point represents three independent trials, 72 < n < 120). B, Stomatal apertures were measured for Col-0, fldh-1, and fldh-2 lines in the presence of 0, 0.5, 2.5, or 5.0 μm cisABA (83 < n < 166). The se of the mean is indicated for all data points. *, **, and *** represent significant differences compared with the Col-0 control of P < 0.05, P < 0.01, and P < 0.001, respectively, as determined by Student’s t test. W/L, Stomatal pore width divided by length.