Role of Farnesyltransferase in ABA Regulation of Guard Cell Anion Channels and Plant Water Loss

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Science  09 Oct 1998:
Vol. 282, Issue 5387, pp. 287-290
DOI: 10.1126/science.282.5387.287


Desiccation of plants during drought can be detrimental to agricultural production. The phytohormone abscisic acid (ABA) reduces water loss by triggering stomatal pore closure in leaves, a process requiring ion-channel modulation by cytoplasmic proteins. Deletion of the Arabidopsis farnesyltransferase geneERA1 or application of farnesyltransferase inhibitors resulted in ABA hypersensitivity of guard cell anion-channel activation and of stomatal closing. ERA1 was expressed in guard cells. Double-mutant analyses of era1 with the ABA-insensitive mutants abi1 and abi2 showed that era1suppresses the ABA-insensitive phenotypes. Moreover, era1plants exhibited a reduction in transpirational water loss during drought treatment.

Protein farnesylation, a posttranslational modification process, mediates the COOH-terminal lipidation of specific cellular signaling proteins, including Ras, guanosine triphosphatases (GTPases), trimeric GTP-binding protein, nuclear lamin B, and yeast mating pheromone a-factor (1). In each of these cases, farnesylation increases membrane association and cellular activity of these proteins. Thus, farnesylation plays an essential role in signal transduction cascades of yeast and mammalian cells (1). In plant cells, farnesyltransferase (FTase) activities have been identified, and changes in FTase activity during cell growth and division have been demonstrated (2, 3). In Arabidopsis, recessive mutations in the ERA1 gene, which encodes the FTase β subunit, were identified and have been shown to prolong seed dormancy due to an enhanced response to ABA (4). This suggests that farnesylation may be essential for negative regulation of ABA signaling in seeds.

Plants lose over 90% of water by transpiration through stomatal pores formed by pairs of guard cells on the leaf surface. The hormone ABA is synthesized in response to drought stress and triggers a signaling cascade in guard cells that results in stomatal closing (5, 6). Studies have indicated that activation of anion channels in the plasma membrane of guard cells is required during ABA-induced stomatal closing (6–8). Coupling of intracellular signaling proteins to membrane ion channels is essential for this ABA-mediated response (6, 8). To investigate whether cytoplasmic regulators are linked to ABA regulation of ion channels by farnesylation, we analyzed the effects of two competitive FTase inhibitors, α-hydroxyfarnesylphosphonic acid (HFPA) (9) and manumycin (10) on ion channels and stomatal movements. Whole cell patch-clamp current recordings (11) showed that, in the presence of ABA, exposure ofArabidopsis guard cells to HFPA significantly increased ABA activation of anion currents (Fig. 1, A and B) (P < 0.004). In the absence of ABA, HFPA did not enhance anion currents (Fig. 1B) (P > 0.5). Stomatal aperture measurements (12) showed that HFPA treatment also increased the ABA sensitivity of stomatal closing (Fig. 1C) (P < 0.001 at 5 μM ABA). ABA-hypersensitive stomatal closure was also detected by use of a different FTase inhibitor, manumycin (n = 3 experiments, 480 stomata).

Figure 1

Protein farnesyltransferase inhibitors affect ABA signaling in Arabidopsis guard cells. (A) The FTase inhibitor HFPA causes increased activation of ABA-regulated anion currents in Arabidopsis guard cells. Guard cells were treated with 10 μM ABA in the absence (–HFPA) or presence (+HFPA) of 2 μM HFPA. Whole cell patch-clamp currents were recorded from a holding potential of +30 mV with membrane voltage steps ranging from –145 mV to +35 mV in +30-mV increments (8). (B) Average magnitudes of steady-state anion currents recorded at –145 mV in the absence and presence of 2 μM HFPA with or without 10 μM ABA. Experiments were performed as in (A). Currents at the end of –145-mV voltage pulses were averaged (n = 10 to 12 guard cells for each condition) (11). (C) HFPA causes increased ABA sensitivity of stomatal closing. Intact leaves were floated in solution with or without 2 μM HFPA under light for 2 hours to induce stomatal opening. Then ABA at indicated concentrations was added to the bath solution to assay stomatal closing (12). Data from four separate experiments (n = 80 stomata per data point) are shown. (D) ERA1 FTase expression patterns in intact leaves of WTArabidopsis and ERA1-promoter–GUS transgenic plants. Transgenic plants contained an ERA1-promoter GUS fusion (ERA1-GUS) and were exposed to blue light. Gene expression resulted in cleavage of the Imagene Green dye to give yellow fluorescence on a red chlorophyll autofluorescent background in intact leaves. Wild-type plants or empty vector–transformed plants were used as controls.

Previous studies have shown that a pea FTase β subunit is expressed in meristematic tissues (13), and mRNA of theArabidopsis ERA1 FTase accumulates in flower buds (4). To determine whether ERA1 is also expressed in guard cells, we analyzed transgenic plants expressingERA1 promoter–GUS constructs in mature leaves (14). In intact leaves, Arabidopsis guard cells showed GUS activities, indicating that the ERA1 gene is expressed in guard cells (Fig. 1D), in addition to expression in other vegetative tissues.

A fast-neutron mutant allele, era1-2, in which the entireERA1 gene is deleted, was used to determine whetherERA1 directly affects guard cell ABA signaling. Stomatal aperture measurements showed that the era1-2 mutation caused ABA hypersensitivity of stomatal closing (Fig. 2A) (P < 0.001 at 10 μM ABA). In the absence of exogenous ABA, stomatal apertures inera1-2 were slightly smaller than those in wild-type (WT) control plants under the imposed conditions (Fig. 2A). When the KCl concentration in solutions and light intensity were increased during stomatal opening, stomata in era1-2 opened as wide as those in WT plants but continued to show ABA hypersensitivity of stomatal closing (15), indicating that smaller stomatal apertures inera1-2 might be due to hypersensitivity to endogenous ABA.

Figure 2

The FTase deletion mutant era1-2causes ABA hypersensitivity of anion-channel activation and of stomatal closing. (A) Comparison of ABA-induced stomatal closing in wild-type (WT) and the era1-2 mutant. Data from three representative (n = 60 stomata per data point) experiments out of nine are shown. (B andC) Whole-cell currents recorded in the absence (–ABA) or presence of 10 μM ABA (+ABA) in WT (B) and in era1-2mutant (C) guard cells. ABA (10 μM) was added to pipette and bath solutions (8). Voltage protocols in (B) and (C) were the same as in Fig. 1A. (D) Steady-state current-voltage relationships show increased ABA activation of anion currents inera1-2 guard cells compared with those in WT guard cells. Recordings were performed as in (B) and (C) (n = 14 to 27 cells averaged per curve). Symbols are as in (B) and (C).

We examined whether the era1 mutation affects ABA regulation of guard cell anion channels (11). In the absence of ABA, era1-2 did not cause constitutive enhancement of anion currents under the imposed conditions (Fig. 2, B and C). In the presence of 10 μM ABA, era1-2 mutation consistently caused increased activation of anion currents compared to WT [Fig. 2, B (n = 30) and C (n = 49)] (16). Current voltage analyses showed that ABA-activated steady-state anion currents were substantially larger inera1-2 than in WT guard cells (Fig. 2D) (P< 0.003 at –145 mV). Interestingly, transient depolarization-activated outward-rectifying K+ currents in the plasma membrane of guard cells were enhanced by the era1mutation in the absence of ABA. For example, peak currents at +100 mV were 254 ± 24 pA (n = 18) in WT and 411 ± 36 pA (n = 15) in era1-2 guard cells (11).

The above data show that deletion of the ERA1 FTase gene causes ABA hypersensitivity of anion-channel activation and of stomatal closing. The findings that FTase inhibitors mimic the ERA1deletion mutation in WT plants (Fig. 1) suggest that ABA hypersensitivity in era1-2 is not due to a long-term effect of FTase deletion during guard cell maturation. Rather, these data suggest that FTases modulate a negative regulation pathway of guard cell ABA signaling.

The ABA-insensitive mutant loci abi1 andabi2 (17) encode type 2C protein phosphatases (PP2C) (18–20). Recent studies have led to models in which these PP2Cs may function as negative regulators in ABA signaling (8, 21). ABI1 and ABI2 do not have farnesylation consensus sequences. To test whether the era1 andabi mutations interact genetically, we generated homozygous double mutants of era1/abi1 and era1/abi2(22). As previously reported, activation of anion currents by ABA is impaired in the abi1 and abi2 mutants (Fig. 3, A and B), consistent with impairment in ABA-induced stomatal closing (8,17, 23). ABA (10 μM) was sufficient to activate anion channel currents in both the era1/abi1 (Fig. 3A) andera1/abi2 double mutants (Fig. 3B). Steady-state current-voltage relations showed that ABA activation of anion currents was restored in these two double mutants (Fig. 3, C and D). Furthermore, ABA-induced stomatal closing was restored in theera1/abi1 double mutant (24) and in theera1/abi2 double mutant (Fig. 3E). Stomatal responses of theera1/abi2 double mutants were similar to those of WT plants, but did not fully show the era1 phenotype. Stomata ofera1/abi1 showed less ABA sensitivity thanera1/abi2, but suppression of the abi1 phenotype was clear (24). The ABA insensitivities of abi1and abi2 in seed germination (17) were also suppressed in these double mutants with a sensitivity sequence ofera1 > era1/abi1era1/abi2 > Ler WT > Col WT >abi1abi2, where Col WT and Ler WT are Colombia WT and Lansberg erecta WT, respectively (25).

Figure 3

ERA1 FTase deletion suppresses the ABA-insensitive (semi-) dominant mutations in era1/abi1and era1/abi2 homozygous double mutants. (A) ABA activation of anion-channel currents was analyzed in the absence (–ABA) or presence of 10 μM ABA (+ABA) in era1/abi1double-mutant guard cells (right) and compared to abi1mutant guard cells (left). (B) ABA activation of anion-channel currents was analyzed in the absence or presence of 10 μM ABA in era1/abi2 double-mutant guard cells and compared to abi2 mutant guard cells. Time and current scale bars refer to both (A) and (B). (C and D) Steady-state current-voltage relationships as recorded in (A) and (B), respectively. Symbols in (C) and (D) correspond to those in (A) and (B), respectively (n = 5 cells for abi1 and abi2;n = 10 to 16 cells for era1/abi1 andera1/abi2 per condition). (E) ERA1 FTase mutation partially suppresses the ABA insensitivity of stomatal closing in theabi2 mutant. ABA-induced stomatal closures inabi2 and era1/abi2 double mutant were compared. Stomatal apertures of abi2, Colombia WT, andera1/abi2 were normalized with respect to the apertures in the absence of ABA, respectively, for comparison; r.u., relative unit. Data from three separate experiments are shown (n = 60 stomata per bar).

Because deletion of the ERA1 FTase potentiates ABA-induced anion currents and stomatal closing in epidermal strips and partially suppresses the abi1 and abi2 mutations, we investigated whether whole plant transpiration is reduced during drought. Both WT plants and era1-2 plants were grown and watered for ≈21 days, and then subjected to drought stress by terminating irrigation (26). Wild-type and era1-2plants showing similar developmental stages and similar number of leaves were specifically selected for drought treatments, and evaporation from soil was minimized by covering the soil in pots. After 12 days of drought treatment, WT plants showed severe wiltiness and chlorosis of rosette leaves. In contrast, era1-2 plants were turgid and leaves remained green (Fig. 4A). Soil water content in pots ofera1-2 plants decreased more slowly during drought stress than those of WT plants (Fig. 4B), consistent with plant phenotypes. The era1-2 plants also showed slowed growth, which may be partially due to increased stomatal closing and reduced carbon fixation, or to ERA1 expression in several vegetative tissues (4, 27), or both. When pots were not covered, the reduced wiltiness of era1-2 plants was visible although less pronounced. Transpiration rates of WT leaves were 2.8 ± 0.3–fold larger than those of era1-2 plants after 10 days of drought (26). Stomatal apertures of both WT and era1-2 decreased during drought (24). However, stomatal apertures of era1-2 decreased faster and were smaller than those of WT during drought (for example, 1.08 ± 0.05 versus 1.24 ± 0.03 μm after 4 to 5 days of drought in noncovered pots; n = 75, P < 0.02). These results show that ERA1 deletion decreases the transpiration rate of leaves and consequently slows desiccation during drought.

Figure 4

Reduced wilting of era1-2 plants during drought stress. (A) Both WT and era1-2plants were grown under normal watering conditions for ∼21 days and then subjected to drought stress by completely terminating irrigation. Pots were covered to minimize soil evaporation. Photo shows four representative plants out of 32 after 12 days of drought stress (26). (B) Changes in soil water content during drought stress treatment of WT and era1-2plants.

Protein farnesylation plays important and diverse roles in cellular processes and signal transduction cascades, which control cell growth, division, morphology, and visual signaling in eukaryotic cells (1–3). Competitive FTase inhibitors, as used here (Fig. 1), reduce Ras-mediated tumor growth (10,28). However, viable null mutants in FTase genes have not yet been found in other multicellular eukaryotes (1), and ion-channel modulation by FTases has not yet been reported. In plants, roles for protein farnesylation have been demonstrated in cell cycle regulation (2, 3, 13) and in seed germination (4). The only plant protein of known function shown to be farnesylated in vivo thus far is ANJ1, which is a homolog of the bacterial molecular chaperone DnaJ (29).

Although era1 affects other signal transduction processes (27), we demonstrate in guard cells a function for protein farnesylation in regulation of ion channels, stomatal movements, and transpirational water loss by modulation of the ABA signaling cascade. Partial suppression of the ABA-insensitive phenotypes of theabi1 and abi2 mutants by ERA1 deletion suggests that the target of the ERA1 FTase may function downstream or parallel to these ABI protein phosphatases. We propose that the ERA1 FTase plays a major role in linking undetermined soluble negative regulatory proteins to plasma membrane ion-channel regulation in guard cells. Modulation of ERA1 or its targets, specifically in guard cells or other cell types, will allow further analysis of ERA1 effects on gas exchange, growth, and development. In conclusion, using several approaches, we provide evidence for a mechanism causing ABA hypersensitivity in guard cell signaling.

  • * To whom correspondence should be addressed at Department of Biology, University of California, San Diego, La Jolla, CA 92093–0116, USA. E-mail: julian{at}


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