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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.
Isoprenylated proteins are modified at the carboxyl terminus by a farnesylcysteine (FC) or geranylgeranylcysteine (GGC) moiety (Clarke, 1992; Zhang and Casey, 1996; Rodríguez-Concepción et al., 1999; Crowell, 2000; Galichet and Gruissem, 2003; Crowell and Huizinga, 2009). These modifications are catalyzed by protein farnesyltransferase (PFT) and protein geranylgeranyltransferase type I (PGGT I), respectively, which transfer the prenyl group from farnesyl diphosphate (FPP) or geranylgeranyl diphosphate (GGPP) to the cysteine residue of a carboxyl terminal CaaX motif (C=Cys; a=aliphatic; X = Cys, Ala, Ser, Gln, Met, Leu, Ile). In a similar manner, protein geranylgeranyltransferase type II (PGGT II) transfers geranylgeranyl groups from geranylgeranyl diphosphate to the carboxyl terminal cysteine residues of RAB proteins bound by the RAB escort protein. Isoprenylated CaaX proteins are subsequently proteolyzed, resulting in the removal of the three amino acids downstream of the isoprenylcysteine residue, and carboxyl methylated. These modifications are catalyzed by CaaX endoproteases and isoprenylcysteine methyltransferases (ICMT), respectively (Crowell et al., 1998; Crowell and Kennedy, 2001; Bracha et al., 2002; Narasimha Chary et al., 2002; Cadiñanos et al., 2003; Bracha-Drori et al., 2008). Demethylation of isoprenylated proteins by isoprenylcysteine methylesterase (ICME) has also been reported (Deem et al., 2006; Huizinga et al., 2008).
In Arabidopsis, protein isoprenylation is involved in negative regulation of abscisic acid (ABA) signaling and meristem development. Loss-of-function mutations in the ENHANCED RESPONSE TO ABA1 (ERA1) gene, which encodes the β-subunit of PFT, cause an enhanced response to ABA and enlarged meristems (Cutler et al., 1996; Pei et al., 1998; Running et al., 1998; Bonetta et al., 2000; Yalovsky et al., 2000; Ziegelhoffer et al., 2000). Consequently, era1 mutants exhibit drought resistance and supernumerary floral organs. Loss-of-function mutations in the PLURIPETALA (PLP) gene, which encodes the α-subunit of both PFT and PGGT 1, also cause an enhanced response to ABA and an exaggerated era1-like developmental phenotype (Running et al., 2004). However, to date, a farnesylated negative regulator of ABA signaling has not been reported. Loss-of-function mutations in the GERANYLGERANYLTRANSFERASE BETA (GGB) gene, which encodes the β-subunit of PGGT I, also cause an enhanced response to ABA in guard cells (but not seeds) and an enhanced response to auxin-induced lateral root formation (Johnson et al., 2005). Consistent with these observations, ROP2 and ROP6 have been identified as negative regulators of ABA signaling (Lemichez et al., 2001; Li et al., 2001; Yang, 2002) and AUX2-11 (IAA4), AGG1, and AGG2 have been identified as negative regulators of auxin-induced lateral root formation (Wyatt et al., 1993; Trusov et al., 2007). All five proteins are predicted or known to be geranylgeranylated (Caldelari et al., 2001; Zeng et al., 2007).
Like all proteins, isoprenylated proteins exist in equilibrium between synthesis and turnover; however, whereas the degradation of unmodified proteins liberates free amino acids, the degradation of isoprenylated proteins also liberates isoprenylcysteine compounds (FC or GGC). Consequently, enzymes exist in eukaryotic cells for the metabolism of FC and GGC. From mammalian systems, it is known that proteolysis of isoprenylated proteins generates FC and GGC, which are metabolized to cysteine and either farnesal or geranylgeranial by prenylcysteine lyase, an FAD-dependent thioether oxidase (Zhang et al., 1997; Tschantz et al., 1999, 2001; Digits et al., 2002). However, prenylcysteines and prenylcysteine methyl esters can also be oxidized by flavin-dependent monooxygenases (FMOs) or P450s, or cleaved by cysteine β-lyases (Cashman et al., 1990; Sausen and Elfarra, 1990; Park et al., 1992, 1994). Prenylcysteine lyase is localized to lysosomal membranes, a major site of lipid metabolism, and catalyzes C–S bond cleavage by a mechanism involving removal of the pro-S hydrogen at the C-1 position of the isoprenyl moiety via sequential one-electron transfers to non-covalently bound FAD. This step is followed by hydrolysis of the resulting thiocarbenium ion to a hemithioacetal intermediate (Tschantz et al., 2001; Digits et al., 2002), which breaks down into cysteine and a prenyl aldehyde. Oxidation of FADH2 by O2 generates H2O2 and completes the catalytic cycle. Although mammalian prenylcysteine lyase is specific for substrates with a primary amino group (i.e. it does not recognize N-acetylated isoprenylcysteine substrates), it lacks significant specificity for the isoprenoid tail (i.e. it exhibits similar kinetics for FC and GGC) (Zhang et al., 1997). Disruption of the prenylcysteine lyase gene in fibroblast cells results in greater sensitivity to growth inhibition in the presence of prenylcysteines (Beigneux et al., 2002), and prenylcysteine lyase knockout mice accumulate significant amounts of FC and GGC in brain and liver tissue. Surprisingly, prenylcysteine accumulation is not accompanied by significant developmental or physiological consequences in these mice (Beigneux et al., 2002).
Farnesal and geranylgeranial metabolism is not well understood, but farnesol and geranylgeraniol metabolism has been analyzed in detail. Isoprenyl alcohols, for example, can be degraded via a mechanism similar to fatty acid metabolism or oxidized at the C-1 and ω positions (Christophe and Popjak, 1961; Havel et al., 1986; Gonzalez-Pacanowska et al., 1988; Keung, 1991; Bostedor et al., 1997; Vaidya et al., 1998; DeBarber et al., 2004). Farnesol and geranylgeraniol can also be sequentially phosphorylated by CTP-dependent kinases and the resulting FPP and GGPP used for isoprenoid biosynthesis (Crick et al., 1997; Westfall et al., 1997; Bentinger et al., 1998; Thai et al., 1999).
Unlike mammals, Arabidopsis plants possess an FC lyase (FCLY) with remarkable specificity for FC (Crowell et al., 2007). Moreover, because the Arabidopsis genome contains no genes with significant relatedness to the FCLY (At5g63910) gene, GGC metabolism is thought to proceed by a different mechanism (e.g. S-oxidation by a flavin-dependent monooxygenase, C–S bond cleavage by a pyridoxal 5-phosphate (PLP)-dependent β-lyase, etc.). Reduction of farnesal to farnesol has been shown to be catalyzed by an NAD(P)H-dependent aldehyde reductase/NAD(P)-dependent alcohol dehydrogenase in Arabidopsis (Crowell et al., 2007). Together, these observations suggest the existence of a recycling pathway in plants whereby the farnesal product of FC lyase is reduced to farnesol, which is subsequently phosphorylated to farnesyl diphosphate. Indeed, CTP-dependent phosphorylation of farnesol to farnesyl diphosphate has been described in plants (Thai et al., 1999). Whereas FC and GGC accumulation results in no apparent phenotype in prenylcysteine lyase knockout mice, fcly-1 and fcly-2 mutants of Arabidopsis, which exhibit 20 and 50% of wild-type FC lyase activity, respectively, are hypersensitive to ABA (Crowell et al., 2007). However, the connection between FCLY function and ABA signaling remains obscure. Because FC compounds are competitive inhibitors of ICMT (Tan et al., 1991; Shi and Rando, 1992; Ma et al., 1995; Narasimha Chary et al., 2002) and ICMT is a negative regulator of ABA signaling (Huizinga et al., 2008), we propose that the hypersensitivity of fcly mutants to ABA is caused by FC accumulation and competitive inhibition of ICMT.
To confirm the selectivity of Arabidopsis FC lyase for FC and gain insights into the catalytic mechanism of this unique enzyme, we performed kinetic analyses on recombinant Arabidopsis FC lyase expressed in Spodoptera frugiperda (Sf9) cells. Furthermore, to confirm or refute the hypothesis that the enhanced response of fcly mutants to ABA is caused by FC accumulation and concomitant inhibition of ICMT, we compared the FC content of wild-type and fcly plants and tested the prediction that overexpression of ICMT would suppress or reverse the ABA phenotype of fcly plants.
To study the kinetics and catalytic mechanism of Arabidopsis FC lyase, a suitable system for expression of recombinant enzyme was identified. We attempted to express the At5g63910 coding sequence in recombinant yeast cells using the pYES2.1/V5-His-TOPO vector, but were unable to detect galactose-inducible FC lyase activity. Consequently, we expressed the At5g63910 coding sequence in Spodoptera frugiperda (Sf9) cells using recombinant baculovirus—a strategy that was used successfully to express human prenylcysteine lyase (Tschantz et al., 1999). As shown in Figure 1, recombinant Arabidopsis FC lyase was successfully expressed in Sf9 cells. The first sample lane of Figure 1 shows a crude lysate from uninfected Sf9 cells. The second and third lanes show supernatant and pellet fractions, respectively, from Sf9 cells 3d post infection (dpi) following lysis in the presence of 0.2% CHAPS and centrifugation at 16000g. A prominent band at approximately 67kDA can be seen in both the detergent soluble and insoluble fractions. Interestingly, the protein is approximately 11.7kDa larger than the predicted molecular mass of the FCLY-encoded protein (55.3kDa)—a difference that agrees almost exactly with the difference between the predicted and apparent molecular masses of human prenylcysteine lyase (Tschantz et al., 1999). In the case of the human enzyme, N-glycosylation was shown to be responsible for this difference (Tschantz et al., 1999). To test the hypothesis that N-glycosylation accounts for the slow migration of recombinant Arabidopsis FC lyase on SDS-polyacrylamide gels, we searched for potential N-glycosylation sites in the Arabidopsis FC lyase coding sequence. As shown in Figure 2, the protein product of the FCLY gene possesses a predicted amino terminal signal sequence and four putative N-glycosylation sites (N-X-S/T), and a Kyte-Doolittle hydropathy plot predicts three or four transmembrane domains (data not shown). In addition, FC lyase synthesized in vitro in the presence of microsomal membranes migrated at a higher apparent molecular mass than FC lyase synthesized in the absence of microsomal membranes (Figure 3). Treatment of the former with endoglycosidase H (Endo H) or peptide N-glycosidase F (PNGase F) caused the protein to co-migrate with FC lyase synthesized in the absence of microsomes, confirming the N-glycosylation of the enzyme (Figure 3). Treatment of recombinant Arabidopsis FC lyase from Sf9 cells with Endo H also caused the enzyme to migrate at an apparent molecular mass of 55kDa (data not shown).
Recombinant Arabidopsis FC lyase activity ([1-3H]FC conversion to [1-3H]farnesal) was linear with time up to 2h in the presence of 5μg protein and linear with the amount of protein in the assay up to 7μg in a 1-h assay (Figure 4). Moreover, the enzyme exhibited saturation kinetics, and a Lineweaver-Burke plot revealed an apparent Km of 45μM for FC and an apparent Vmax of 417pmolmin−1mg−1. To test the substrate specificity of recombinant Arabidopsis FC lyase, the ability of unlabeled FC, GGC, and other prenylcysteine analogs to inhibit the conversion of [1-3H]FC to [1-3H]farnesal was measured. As shown in Figure 5 and Table 1, unlabeled FC effectively competed with [1-3H]FC and exhibited an IC50 of 50μM. Substrate analogs with alterations on the amino acyl side of the sulfur atom (N-acetyl FC and farnesyl homocysteine) also competed, but with IC50 values 4–10-fold higher than that of unlabeled FC. In contrast, analogs with alterations in the prenyl moiety were significantly less competitive. In fact, geranylgeranylcysteine, nerylcysteine, citronellylcysteine, and benzylcysteine did not compete (no inhibition of [1-3H]FC oxidation was observed at concentrations as high as 500μM). The exception to this was geranylcysteine, which exhibited an IC50 four-fold higher than that of unlabeled FC. These data demonstrate the specificity of Arabidopsis FC lyase, which appears to have no detectable activity toward geranylgeranylated substrates, and support the hypothesis that FC and GGC metabolism proceed by different mechanisms. Further support for this hypothesis can be found in Supplementary Data, where it is shown that [1-3H]GGC metabolism does not involve a geranylgeranial intermediate.
As shown in Table 1, diphenyl iodonium inhibited the FC lyase reaction. This observation suggests that, like mammalian prenylcysteine lyase, Arabidopsis FC lyase is a flavoprotein (Coves et al., 1999; Shiemke et al., 2004). To further examine the mechanism of action of Arabidopsis FC lyase, its dependence on FAD and molecular oxygen was tested. As shown in Figure 6, the addition of excess FAD to the reaction enhanced the FC lyase reaction by 30% (reactions were performed at substrate saturation as described in Methods). This result is consistent with the hypothesis that FAD is tightly, but non-covalently, bound to FC lyase and is required for FC lyase activity. To determine whether Arabidopsis FC lyase requires molecular oxygen, experiments were performed to monitor [1-3H]FC metabolism under anaerobic and aerobic (control) conditions. As expected, degassing the buffer solution caused a significant decrease in FC lyase activity (Figure 6). Moreover, the addition of glucose and glucose oxidase, which catalyzes the O2-dependent oxidation of β-D-glucose to D-glucono-1,5-lactone and H2O2, caused a 46% decrease in FC lyase activity, and the addition of glucose, glucose oxidase, and catalase caused a 66% decrease in activity (the latter prevents product inhibition of glucose oxidase by converting H2O2 to H2O and O2; however, two molecules of O2 are consumed by glucose oxidase for every molecule of O2 regenerated by catalase). These results support the mechanism proposed by Tschantz et al. (2001) for enzymes in the prenylcysteine lyase family. Hydrogen peroxide production by FC lyase was detected spectrophotometrically at 405nm using ABTS (2,2′-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid)) and horseradish peroxidase and was slightly less than stoichiometric relative to farnesal production, presumably due to the presence of a peroxidase activity in the membrane fraction of recombinant Sf9 cells (Table 2).
As stated above, fcly mutants of Arabidopsis exhibit reduced FC lyase activity and an enhanced response to ABA (Crowell et al., 2007). One hypothesis to explain this phenotype is that FC accumulation in fcly plants leads to competitive inhibition of ICMT activity. Because ICMT is a negative regulator of ABA signaling (Huizinga et al., 2008), it follows that inhibition of ICMT would result in an enhanced response to ABA. To test this hypothesis, Col-0, era1-2, fcly-1, and fcly-2 plants were analyzed for FC accumulation. era1-2 plants were included in this experiment as a negative control and were expected to contain little or no FC (i.e. because era1-2 plants lack the β-subunit of PFT). As shown in Figure 7, extracts of era1-2 plants contained significantly lower levels of FC than extracts of Col-0 plants (normalized to chlorophyll a, which co-extracts with FC). That era1-2 plants contained measurable amounts of FC, albeit greatly reduced, confirms the isoprenoid cross-specificity of PFT and PGGT1 and provides an explanation for the partial compensation of the PFT defect in era1-2 plants by PGGT1 (i.e. PGGT1 has a weak protein farnesyltransferase activity) (Trueblood et al., 1993; Yokoyama et al., 1995; Johnson et al., 2005). fcly-1 and fcly-2 plants, on the other hand, contained significantly higher levels of FC than Col-0 plants (Figure 7). fcly-1, which exhibits 20% of wild-type FC lyase activity (Crowell et al., 2007), contained 30% more FC than Col-0, whereas fcly-2, which exhibits 50% of wild-type FC lyase activity (Crowell et al., 2007), contained 11% more FC than Col-0. Similar results were obtained when the data were normalized to fresh weight.
The next test was to determine whether overexpression of AtSTE14B (ICMT) suppresses or reverses the ABA phenotype of fcly-1 and fcly-2 plants—a result that would be expected if the ABA hypersensitivity of fcly-1 and fcly-2 plants was the result of competitive inhibition of ICMT by accumulated FC (FC is a substrate of ICMT and competes with prenylated proteins for the ICMT active site; Tan et al., 1991; Shi and Rando, 1992; Ma et al., 1995; Narasimha Chary et al., 2002). As shown in Figure 8, ICMT overexpression was achieved in both fcly-1 and fcly-2 plants. These lines, called fcly-1:ICMT°x and fcly-2:ICMT°x, exhibited significantly increased ICMT activity compared to the corresponding fcly-1 and fcly-2 lines. As shown in Figure 9, both fcly-1 and fcly-2 exhibited an enhanced response to ABA-induced inhibition of seed germination. As expected, fcly-1 exhibited the more dramatic phenotype. Overexpression of ICMT suppressed the ABA hypersensitivity of the fcly-1 line and, consequently, germination of fcly-1:ICMT°x seeds was identical to that of wild-type Col-0 seeds (Figure 9). Interestingly, overexpression of ICMT in the fcly-2 line effectively reversed the ABA hypersensitive phenotype and caused an ABA-insensitive phenotype in fcly-2:ICMT°x seeds (Figure 9). This observation is most likely due to two factors: (1) the relatively modest ABA hypersensitivity of fcly-2 seeds, and (2) the strong overexpression of ICMT achieved in the fcly-2:ICMT°x line.
To further examine the effect of ICMT overexpression on the response of fcly-1 and fcly-2 plants to ABA, we examined ABA-induced stomatal closure in Col-0, fcly-1, fcly-2, fcly-1:ICMT°x, and fcly-2:ICMT°x plants. As shown in Figure 10, fcly-1 and fcly-2 plants exhibited an enhanced response to ABA-induced stomatal closure. However, overexpression of ICMT caused a decreased response to ABA in fcly-1:ICMT°x and fcly-2:ICMT°x guard cells. These results, together with the results shown in Figures 7 and and9,9, provide strong support for the hypothesis that the enhanced response of fcly-1 and fcly-2 plants to ABA is due to FC accumulation and competitive inhibition of ICMT. Further support for the hypothesis that FC competes with ICMT substrates can be found in Supplementary Data, where it is shown that FC competitively inhibits ICMT-mediated methylation of N-acetyl-S-trans, trans-farnesyl-L-cysteine with an apparent IC50 of 50μM.
In this paper, recombinant Arabidopsis FC lyase expressed in Sf9 cells is shown to migrate by SDS–PAGE at an apparent molecular mass approximately 11.7kDa greater than its predicted molecular mass (Figure 1). This difference was recapitulated by in vitro translation of Arabidopsis FC lyase in the presence or absence of microsomal membranes—an observation that strongly suggests the enzyme is proteolytically processed at the amino terminus and post-translationally N-glycosylated (Figure 3). Treatment with Endo H or PNGase F caused FC lyase translated in the presence of microsomal membranes, or expressed in Sf9 cells, to co-migrate with FC lyase translated in the absence of microsomes, supporting the hypothesis that it is N-glycosylated (Figure 3). Recombinant Arabidopsis FC lyase exhibited classical Michaelis-Menten kinetics, with an apparent Km for FC of 45μM (Figure 4). Moreover, the enzyme selectively oxidized FC, and exhibited a requirement for FAD and molecular oxygen (Table 1 and Figure 6). fcly-1 and fcly-2 mutant plants, which possess approximately 20 and 50% of wild-type FC lyase activity, respectively (Crowell et al., 2007), accumulated significant amounts of FC compared to wild-type plants and exhibited a greater response to ABA in seed germination and stomatal closure assays (Figures 7, ,9,9, and and10).10). Overexpression of ICMT suppressed the enhanced response of fcly-1 seeds to ABA and reversed the enhanced response of fcly-2 seeds to ABA (Figure 9). Moreover, the ABA hypersensitivity of fcly-1 and fcly-2 stomata was reversed by ICMT overexpression (Figure 10). These and other data (Crowell et al., 2007) demonstrate that, like ICMT, FCLY is a negative regulator of ABA signaling.
The enhanced response of fcly mutants to ABA could be due to FC over-accumulation and competitive inhibition of ICMT, which would be expected to cause incomplete processing of prenylated proteins, or farnesal and farnesol under-accumulation, which would potentially cause reduced protein farnesylation. The data presented here favor the former hypothesis because: (1) FC accumulates in fcly mutant plants, and (2) overexpression of ICMT suppresses or reverses the ABA phenotype of fcly plants (i.e. ICMT overexpression would not reverse the effects of decreased protein farnesylation). Nevertheless, it is surprising that the modest accumulation of FC observed in fcly mutants results in significant inhibition of ICMT. This observation suggests that prenylated protein substrates do not effectively compete with excess FC for the ICMT active site in fcly-1 and fcly-2 mutants. Furthermore, loss of FC lyase function may cause FC to accumulate in the endoplasmic reticulum, where ICMT is located.
In mammalian cells, prenylcysteine lyase is localized to lysosomes. This is an interesting observation in view of the fact that the enzyme generates hydrogen peroxide (i.e. the enzyme is not localized to peroxisomes where hydrogen peroxide can be metabolized). FC lyase is similarly localized in Arabidopsis and has been detected in proteomic analyses of vacuolar proteins (Shimaoka et al., 2004; Jaquinod et al., 2007). In addition to a predicted amino terminal signal sequence, the FCLY coding region also contains two potential sequence-specific vacuolar sorting signals [N/L]-[P/I/L]-[I/P]-[R/N/S] (Carter et al., 2004; Jolliffe et al., 2005; Vitale and Hinz, 2005), one of which is near the carboxyl terminus of the protein. In contrast, neither of two possible peroxisome targeting signals is found on the FCLY protein (PTS1 consists of the carboxyl terminal tripeptide [S/C/A]-[K/R]-[L/M/I/F], whereas PTS2, which is typically found within 30 amino acids of the amino terminus, consists of the nonapeptide R-[X]6-[H/K]-[L/I/F] (Hayashi and Nishimura, 2003; Baker and Sparkes, 2005)).
Arabidopsis FC lyase is unique in many respects. First, reduced FC lyase activity causes a measureable phenotype in fcly-1 and fcly-2 mutants of Arabidopsis. Second, unlike the mammalian ortholog, Arabidopsis FC lyase specifically oxidizes FC (Zhang et al., 1997; Crowell et al., 2007). Why the plant enzyme would have evolved to exhibit specificity for FC is a matter of conjecture, but perhaps geranylgeranial and geranylgeraniol are more toxic to plant cells than farnesal and farnesol, which would result in evolutionary pressures to metabolize GGC by an alternative pathway. Consistent with the hypothesis that FC and GGC are metabolized by different mechanisms, we detected no aldehyde product of [1-3H]GGC in the presence of Arabidopsis membranes (i.e. no metabolite was detected that co-migrated by either HPLC or GC with geranylgernial, see Supplementary Data). Rather, we detected three metabolites, one of which co-migrated by HPLC with GGC sulfoxide, but the identities of these metabolites have yet to be definitively established.
Isoprenylcysteine methylation and demethylation were recently shown to regulate ABA signaling in Arabidopsis (Huizinga et al., 2008). ICMT overexpressing lines of Arabidopsis exhibited an ABA-insensitive phenotype in seed germination and stomatal closure assays, whereas ICME overexpressing lines exhibited an ABA-hypersensitive phenotype in these assays. ABA was also shown to induce the expression of the ICME gene (Huizinga et al., 2008). Thus, like ABA biosynthesis, ABA signaling is under positive feedback regulation (i.e. ABA increases ABA sensitivity of plant cells via induction of ICME expression and demethylation (i.e. inactivation) of isoprenylated negative regulators of ABA signaling). The balance between methylation and demethylation of isoprenylated negative regulators of ABA signaling can be perturbed in a number of ways. In this report, it is shown that this balance is perturbed in fcly mutants by the accumulation of FC, a competitive inhibitor of ICMT (Tan et al., 1991; Shi and Rando, 1992; Ma et al., 1995; Narasimha Chary et al., 2002). Consequently, in fcly plants, decreased isoprenylcysteine methyltransferase function results in an enhanced response to ABA. This is unlike the situation in mice lacking a functional prenylcysteine lyase gene because, despite the accumulation of FC and GGC in these mice, no developmental or pathological defects were observed (Beigneux et al., 2002). This observation is unexpected because icmt–/– knockout mice do not survive (Bergo et al., 2001). Thus, it is reasonable to speculate that the accumulated FC and GGC in prenylcysteine lyase knockout mice must be sequestered in an intracellular compartment that prevents inhibition of ICMT.
Surface sterilization of Arabidopsis (Arabidopsis thaliana) seeds was accomplished using the following protocol: 95% ethanol for 5min, 20–50% bleach for 5–20min, five washes in sterile de-ionized water. Sterile seeds were suspended in 0.1% agar, stratified on 1/2X Murashige-Skoog (MS) plates containing 1% sucrose and 0.8% agar for 3d at 4°C, and germinated at 22°C under long day conditions (18h of white light at 100μmolm−2sec−1 followed by 6h of dark) in a vertical orientation. Seedlings were harvested after 4d for extraction of membranes or transferred to ProMix and propagated under the same conditions for analysis of stomatal function or collection of seed. Plants were fertilized with a standard mixture of macro- and micro-nutrients from below.
The FCLY cDNA sequence was PCR-amplified using the primers 5′-AGA TCT ATG AAA GAT TTC CCG ATA GCA ATC TC-3’ (containing a Bgl11 site) and 5′-GGA TCC TTA TGA GTC TGA GTG CAG ACC AGA A-3’ (containing a BamH1 site), inserted into the pCR2.1–TOPO vector, and subjected to DNA sequence analysis. The 1.5-kbp FCLY cDNA was then recloned into the Bgl11 and BamH1 sites of the Baculovirus transfer vector pVL1392, confirmed by DNA sequence analysis, and transfected into Sf9 insect cells using the BD BaculoGold kit (BD Biosciences/Pharmingen, San Diego, CA). Recombinant baculovirus expressing FCLY was amplified using the supplier's protocol. A 30-ml suspension culture of Sf9 cells (106cellsml−1 in Grace's Medium supplemented with 10% fetal bovine serum) was infected with 1ml of recombinant baculovirus (107–108pfuml−1). Cells were then incubated on a rotary shaker at 100rpm for 3d at room temperature. At 3d post infection (3dpi), cells were sedimented at 3000g and re-suspended in 3ml of 40mM Tris-HCl, pH7.5, 1mM DTT containing Complete® protease inhibitors (Roche Diagnostics, Indianapolis, IN). The suspension was then homogenized on ice for 3min and centrifuged at 16000g for 10min. The resulting supernatant was centrifuged at 100000g for 1h and membranes were re-suspended in 300μl of 40mM Tris-HCl, pH7.5, 1mM DTT, 15% glycerol and stored at –80°C.
FC lyase reactions were performed in a total volume of 20μl and consisted of 5μl of membrane protein from recombinant Sf9 cells (0–15μg of protein) and 0–100μM [1-3H]farnesylcysteine (12.5Cimmol−1) in 40mM Tris-HCl, pH7.5. One or more of the following were added, as required: 5mM FAD, 20mM glucose, 2U glucose oxidase, 11U catalase. To remove oxygen from the system, 40mM Tris-HCl, pH7.5, was repeatedly frozen and thawed under vacuum, sparged with nitrogen gas, and FC lyase reactions were started under a nitrogen atmosphere. Reactions were incubated at 30°C for various times (0–6h), terminated with 5μl of methanol, and a 10-μl portion was analyzed, along with authentic farnesol and farnesal standards, by thin layer chromatography using a silica gel plate and hexane:tetrahydrofuran (3:1) as a mobile phase. TLC plates were cut into six sections and analyzed by liquid scintillation. Alternatively, the assay mixture was diluted with 100μl of water and extracted with 200μl of hexane, after which the organic and aqueous layers were partitioned and analyzed by liquid scintillation. Radioactivity detected by liquid scintillation was multiplied by 2 before calculating enzyme activity because FC lyase is stereospecific at the C-1 position, whereas [1-3H]FC is racemic. Consequently, only 50% of the reaction products were expected to be radioactive.
Unlabeled FC and prenylcysteine analogs were synthesized according to published procedures (Brown et al., 1991) from cysteine or homocysteine and the appropriate prenyl halides, which were prepared from the corresponding prenyl alcohols (Aldrich) by sequential mesylation (MsCl, Et3N) and reaction with LiCl or by treatment with phosphorus tribromide (PBr3). Farnesal and geranylgeranial standards were synthesized by Dess-Martin periodinane oxidation of farnesol and geranylgeraniol, respectively. GGC sulfoxide was prepared by treatment of GGC with hydrogen peroxide as described (Cashman et al., 1990).
Extracts of infected Sf9 cells (106cellsml−1, 4dpi) were prepared by centrifugation of infected cells at 3000g, after which the cell pellet was lysed in 40mM Tris-HCl, pH7.5, containing 1% Triton X-100, 150mM NaCl and Complete® protease inhibitors (Roche Diagnostics, Indianapolis, IN). After standing on ice for 30min, the mixture was centrifuged at 16000g for 10min and the supernatant was used to analyze the production of farnesal and hydrogen peroxide by recombinant Arabidopsis FC lyase (to account for product reduction, farnesal and farnesol were quantified by GC and summed). Peroxide formation was determined spectrophotometrically at 405nm using ABTS (2,2′-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid)) and horseradish peroxidase.
In vitro transcription/translation using a reticulocyte-based translation system and canine pancreatic microsomal membranes is a common method for demonstrating in vitro N-glycosylation of plant proteins (Dellapenna and Bennett, 1988; Devoto et al., 1999; Abell et al., 2002; McCartney et al., 2004). In vitro transcription/translation was performed in the presence of [35S]methionine (10mCiml−1, 1000Cimmol−1, PerkinElmer, Boston, MA) using the TnT Quick Coupled Transcription/Translation System (Promega, Madison, WI) and canine pancreatic microsomal membranes (Promega, Madison, WI) according to the manufacturer's specifications. Endoglycosidase H and peptide N-glycosidase F were from Sigma-Aldrich (St Louis, MO) and were used according to the manufacturer's instructions following solubilization of microsomal membranes in 0.2% SDS.
Arabidopsis seedlings were pulverized in liquid nitrogen and stored at –80°C. Pulverized seedlings were then placed in a mortar and pestle and homogenized with 3mL of cold ethanol. The material was transferred to a conical vial, sonicated (12W, 1min), filtered, and the solvent was removed under reduced pressure. The resulting residue was dissolved in 300μL of ethanol and a portion (25μL) analyzed for chlorophyll content (i.e. diluted to 1mL in ethanol, and absorbances determined at 464 and 646nm). To the remainder was sequentially added 450μL of water, 60μL of pyridine, and 100μL of ethyl chloroformate. The mixture was vortexed and allowed to stand for 15min at room temperature, at which time it was extracted three times with 500μL of dichloromethane. The organic extracts were combined, filtered through a short silica gel pad, and concentrated. The residue was then re-dissolved in isopropanol (50–100μL) and subjected to GC analysis (CP-Sil 19 CB column, Varian) using the following GC conditions: flow rate=1.5mLmin−1; column temperature=150°C, 1min; 15°Cmin−1 to 250°C; 250°C, 20min. Under these conditions, the N-ethoxycarbonyl derivative of FC and the N-ethoxycarbonyl ethyl ester of FC eluted at 13.0 and 13.75min, respectively. FC levels were quantified by correlation with a GC calibration curve using a synthesized FC derivative.
To generate the fcly-1:ICMT°x and fcly-2:ICMT°x lines, fcly-1 and fcly-2 mutants of Arabidopsis were transformed with a recombinant binary vector (pCL108) containing the CaMV 35S promoter and the AtSTE14B (ICMT, At5g08335) coding sequence (Huizinga et al., 2008). Agrobacterium tumefaciens strain GV3101 (pMP90) (Koncz and Schell, 1986) and the floral dip method (Clough and Bent, 1998) were used for plant transformation. T1 transformants were selected on 1/2X Murashige-Skoog plates containing 1% sucrose, 0.8% agar, and 200μgml−1 gentamycin. Transgenic plant lines were propagated to the T3 generation and scored for homozygosity by PCR and non-segregation of the antibiotic resistance marker.
Four-day-old Arabidopsis seedlings were pulverized in a mortar and pestle at 4°C in homogenization buffer containing 50mM Hepes, pH7.4, 500mM mannitol, 5mM EDTA, 5mM DTT, and Complete® protease inhibitors (Roche Diagnostics, Indianapolis, IN). Extracts were filtered through cheesecloth, centrifuged at 8000g for 10min at 4°C, and membranes were sedimented from extract supernatants at 100000g for 60min at 4°C. Membranes were then re-suspended in a buffer containing 2.5mM Hepes, pH7.0, 250mM mannitol, and 1mM DTT and aliquots were stored in the presence of 15% glycerol at −80°C. ICMT assays were performed as described (Crowell et al., 1998) in the presence of 100mM Hepes, pH7.0, 60μg of membrane protein, 24μM (2.5Cimmol−1, 60μCiml−1) S-adenosyl-L-[3H methyl]methionine as a methyl donor (GE Healthcare Life Sciences, Piscataway, NJ), and 200μM AFC (BioMol, Plymouth Meeting, PA) as a methyl acceptor in a total volume of 50μl. Reactions were incubated at 30°C for 10–40min, terminated by the addition of 50μl of 90% v/v methylene chloride, 9.75% v/v methanol, 0.25% v/v acetic acid, mixed vigorously, and centrifuged in a microcentrifuge for 1min. A 10-μl portion of the organic phase was spotted onto a plastic-backed silica gel plate and developed in 90% methylene chloride, 9.75% methanol, 0.25% acetic acid. Plates were then sprayed with En3hance fluorographic reagent (NEN/DuPont, Boston, MA) and fluorography was performed at –80°C using X-Omat AR film (Eastman Kodak, Rochester, NY). Liquid scintillation of excised TLC spots was performed to quantify ICMT activity.
Seeds used for germination assays were harvested from control and experimental plants, which were grown together under identical conditions. Seeds were surface-sterilized, suspended in sterile 0.1% agar, and placed on 1/2X Murashige-Skoog plates containing 1% sucrose and 0.8% agar in the dark at 22°C (Cutler et al., 1996). Seeds from control and experimental plants were sown on the same plates and germination (radical emergence) was scored in the presence of various concentrations of exogenous ABA under a dissecting microscope.
Mature rosette leaves were excised and incubated in the presence of various concentrations of ABA or an equivalent volume of DMSO (as a solvent control) in 10ml of water for 2h. Abaxial epidermal peels were then prepared as described (Huizinga et al., 2008), stained with toluidine blue, mounted on a microscope slide, and visualized with a Nikon Eclipse E600 microscope interfaced to a SPOT digital camera. In random order, K.G.K. performed leaf excision and incubation in the presence of various concentrations of ABA. D.H.H. prepared epidermal peels and performed photography and measurement of stomatal apertures without knowledge of sample identities. Data are recorded as the average width/length of individual apertures (n=50 per sample).
Throughout this paper, data are presented as the mean plus or minus the standard error of the mean. Unlike the standard deviation, the standard error of the mean takes into account sample size and is, thus, a more meaningful measure of error. In all data figures, experimental plants are compared to the Col-0 control at the same time and/or concentration of ABA. Because these are pair-wise comparisons, a student's t-test was performed to assess the statistical significance of these differences and only differences with a P-value less than or equal to 0.05 (95% confidence) were considered to be significant.
Supplementary Data are available at Molecular Plant Online.
This work was supported by NSF grant MCB-0900962. The authors also acknowledge NIH Grant P20RR16454 from the INBRE program of the National Center for Research Resources, which provided funds for the Molecular Research Core Facility at Idaho State University, and the Mentored Undergraduate Summer Experience (MUSE) program at The College of New Jersey.
No conflict of interest declared.