Farnesylcysteine lyase is involved in negative regulation of abscisic acid signaling in Arabidopsis

Abstract

The Arabidopsis FCLY gene encodes a specific farnesylcysteine (FC) lyase, which is responsible for the oxidative metabolism of FC to farnesal and cysteine. In addition, fcly mutants with quantitative decreases in FC lyase activity exhibit an enhanced response to ABA. However, the enzymological properties of the FCLY-encoded enzyme and its precise role in ABA signaling remain unclear. Here, we show that recombinant Arabidopsis FC lyase expressed in insect cells exhibits high selectivity for FC as a substrate and requires FAD and molecular oxygen for activity. Arabidopsis FC lyase is also shown to undergo post-translational N-glycosylation. FC, which is a competitive inhibitor of isoprenylcysteine methyltransferase (ICMT), accumulates in fcly mutants. Moreover, the enhanced response of fcly mutants to ABA is reversed by ICMT overexpression. These observations support the hypothesis that the ABA hypersensitive phenotype of fcly plants is the result of FC accumulation and inhibition of ICMT.

Figure 1.

Expression of Recombinant Arabidopsis FC Lyase in Insect Cells. MW, MW markers are indicated; Sf9, crude extract from uninfected Sf9 cells; S, supernatant fraction from Sf9 cells infected with FCLY-recombinant virus (3 dpi) following lysis in the presence of 0.2% CHAPS and centrifugation at 16 000 g; P, pellet fraction from Sf9 cells infected with FCLY-recombinant virus (3 dpi) following lysis in the presence of 0.2% CHAPS, centrifugation at 16 000 g, and solubilization of the pellet in 0.5% TritonX-100. The predicted molecular mass of Arabidopsis FC lyase is 55.3 kDa (see arrow). Recombinant Arabidopsis FC lyase from insect cells migrates at approximately 67 kDa, suggesting possible post-translational N-glycosylation.

Figure 2.

Features of the FCLY Coding Sequence. The FCLY coding sequence is predicted to encode a protein with an amino terminal signal peptide and four potential N-glycosylation sites. The arrow represents the probable site of signal peptide cleavage and asterisks mark the positions of putative N-glycosylation sites. A potential sequence-specific vacuolar-sorting signal near the carboxyl terminus is underlined.

Figure 3.

N-Glycosylation of Arabidopsis FC Lyase In Vitro. Arabidopsis FC lyase was synthesized in vitro using a TnT transcription/translation kit (Promega) in the presence or absence of canine pancreatic microsomal membranes and analyzed by SDS–PAGE and autoradiography. Following in vitro translation in the presence of microsomal membranes, samples were either analyzed directly or solubilized with 0.2% SDS, treated with Endo H or PNGase F, and analyzed. An unprogrammed translation reaction lacking FCLY DNA was also analyzed.

Figure 4.

Michaelis-Menten Kinetics of Recombinant Arabidopsis FC Lyase Expressed in Sf9 Cells. FC lyase reactions were carried out as described in the Methods section. The standard error of the mean is indicated in (A)–(D); however, the errors bars are obscured by the plot symbols in (C) and (D). (A) Linearity of FC lyase activity with time. Reactions contained 5 μg of protein and 5 μM [1-³H]FC. (B) Linearity of FC lyase activity with protein. Reactions contained 4 μM [1-³H]FC and were incubated for 1 h. (C) Substrate saturation kinetics of the FC lyase reaction. Reactions contained 3 μg of protein and were incubated for 1 h. (D) Lineweaver-Burke plot of FC lyase substrate saturation kinetics. The data in Figure 4C were used for Lineweaver-Burke analysis.

Figure 5.

Structures of the Prenylcysteine Analogs used in Table 1.

Figure 6.

Dependence of Arabidopsis FC Lyase on FAD and Molecular Oxygen. FC lyase reactions were performed as described in the Methods section (3.5 μg protein, 4.5 μM [1-³H]FC, incubation for 0.5 or 1 h) in the presence of 5 mM FAD, degassed buffer, 2 U glucose oxidase, 2 U glucose oxidase plus 11 U catalase, and 11 U catalase. 20 mM glucose was added to all reactions containing glucose oxidase. * and *** represent significant differences compared with the control of P < 0.05 and P < 0.001, respectively, as determined by student's t-test.

Figure 7.

Quantification of FC Accumulation in Col-0, era1-2, fcly-1, and fcly-2 Seedlings. The standard error of the mean is shown. * and *** represent significant differences compared with the Col-0 control of P < 0.05 and P < 0.001, respectively, as determined by student's t-test.

Figure 8.

ICMT Activity in Col, fcly-1, fcly-2, fcly-1:ICMT°^x, and fcly-2:ICMT°^x Lines of Arabidopsis. ICMT activity is shown as a function of time for Col, fcly-1, fcly-2, fcly-1:ICMT°^x, and fcly-2:ICMT°^x (membranes were prepared from approximately 100 seedlings for each sample). Assays were performed as described in the Methods section. ICMT activity is indicated as the mean plus or minus the standard error of the mean (n  =  4).

Figure 9.

Overexpression of ICMT Suppresses or Reverses the Enhanced Response of fcly-1 and fcly-2 Seeds to ABA. Seed germination was scored for Col-0, fcly-1, fcly-2, fcly-1:ICME°^x, and fcly-2:ICME°^x lines as a function of time in the presence of 0, 0.3, 1.0, or 5.0 μM cisABA (each data point represents four independent trials, 65 < n < 101). The standard error of the mean is indicated for all data points; for most, the error bars are hidden by the symbols. * and ** represent significant differences compared with the Col-0 control of P < 0.05 and P < 0.01, respectively, as determined by student's t-test. (A) Germination of Col and fcly-1 seeds. (B) Germination of Col and fcly-2 seeds. (C) Germination of Col and fcly-1:ICMT°^x seeds. (D) Germination of Col and fcly-2:ICMT°^x seeds.

Figure 10.

Overexpression of ICMT Reverses the Enhanced Response of fcly-1 and fcly-2 Stomata to ABA. Stomatal apertures were measured for Col-0, fcly-1, fcly-2, fcly-1:ICMT°^x, and fcly-2:ICMT°^x lines after incubation of excised leaves for 2 h in the presence of 0, 0.5, or 2.5 μM cisABA. Stomatal apertures were measured from photomicrographs of epidermal peels and reported as the average of 50 stomata per data point. The standard error of the mean is shown. *, **, and *** represent significant differences compared with the Col-0 control of P  = 0.05, P < 0.05, and P < 0.01, respectively, as determined by student's t-test.