FERONIA receptor kinase pathway suppresses abscisic acid signaling in Arabidopsis by activating ABI2 phosphatase

Abstract

Plant growth and development are controlled by a delicate balance of hormonal cues. Growth-promoting hormones and growth-inhibiting counterparts often antagonize each other in their action, but the molecular mechanisms underlying these events remain largely unknown. Here, we report a cross-talk mechanism that enables a receptor-like kinase, FERONIA (FER), a positive regulator of auxin-promoted growth, to suppress the abscisic acid (ABA) response through activation of ABI2, a negative regulator of ABA signaling. The FER pathway consists of a FER kinase interacting with guanine exchange factors GEF1, GEF4, and GEF10 that, in turn, activate GTPase ROP11/ARAC10. Arabidopsis mutants disrupted in any step of the FER pathway, including fer, gef1gef4gef10, or rop11/arac10, all displayed an ABA-hypersensitive response, implicating the FER pathway in the suppression mechanism. In search of the target for the FER pathway, we found that the ROP11/ARAC10 protein physically interacted with the ABI2 phosphatase and enhanced its activity, thereby linking the FER pathway with the inhibition of ABA signaling.

Fig. 1.

Hypersensitive ABA responses of fer mutant plants. (A–C) ABA sensitivity in seedlings. Approximately 150 seeds from four independent seed lots of simultaneously grown WT (Columbia-0 and C24), fer-4, fer-5, and srn mutants were sown on agar plates supplemented with ABA (mixed isomers; Sigma-Aldrich A1049). (A) The images of plants were taken on day 9 after germination on MS agar medium supplemented with 0 μM (a) or 0.25 μM ABA (b). (B) Close-up images show Col-0 WT (Top), fer-4 (Middle), and fer-5 (Bottom) seedlings taken from Ab. (C) Images indicate srn mutant and WT C24 plants on day 8 after germination on MS agar medium supplemented with 0 μM (MS) or 0.5 μM ABA (MS+ABA). (D) ABA-induced growth inhibition in roots. Five-day-old seedlings grown on the MS medium were transferred to MS medium containing 3 μM ABA. Root length was measured 4 d after transfer. Each data point is the average ± SE of three independent experiments with 10 plants each. (E) Stomatal aperture before and after ABA treatment of fer-4 and WT leaves. Data are presented as average ± SE of three replicates with 10 apertures each. Three independent experiments yielded similar results. (F) Quantification of ROS production in guard cells of the WT and fer-4 plants after ABA treatment. Confocal fluorescence intensities were quantified as average pixel intensities in three random regions of each guard cell by using the OLYMPUS FV1000 software. The relative ROS production of each treatment was normalized to untreated WT (100%). Data are average values ± SE of nine guard cells per genotype in one experiment. Four independent experiments were conducted with similar results.

Fig. 2.

ROP11/ARAC10 interactions with GEFs and ABI2 and expression patterns of ROP11/ARAC10, GEF1, and FER. (A) Y2H assays showing WT or CA-ROP11/ARAC10, but not DN-ROP11/ARAC10, interacting with ABI2. (B) DN-ROP11/ARAC10 interacts with full-length GEF1, GEF4, and GEF10 in the Y2H assays. (C) ROP11/ARAC10 and ABI2 interacted in BiFC assays in Arabidopsis protoplasts. (a) Interacting nVenus–ROP11/ARAC10 and ABI2–cCFP was observed as GFP image. (d) Negative control (nVenus–ROP11/ARAC10 with cCFP vector) was shown. (b and e) Chloroplasts are indicated by red auto-fluorescence. (c and f) Merged images of GFP and red signals are shown. (Scale bars: 10 μm.) (D a–d) The expression of ROP11/ARAC10–GUS reporter in leaves (a), guard cells (b), roots (c), and pollen (d). (e–g) GEF1–GUS expression was also detected in leaves (e), guard cells (f), and roots (g). (h) ProFER-FER-GFP was expressed in epidermal and guard cells. (Scale bars: b–d, f, and g, 20 μm; a and e, 0.5 cm).

Fig. 3.

ROP11/ARAC10 activates ABI2 phosphatase, and rop11/arac10 mutants are ABA-hypersensitive. (A) ROP11/ARAC10 enhances phosphatase activity of ABI2. Phosphopeptide phosphatase assays were conducted as described in Materials and Methods. Data are presented as average activity ± SE of three replicates in one experiment. Three independent experiments were conducted with similar results. (B) OST1 kinase activity was inhibited by ROP11/ARAC10–ABI2. Equal amount of OST1 was added to individual kinase reactions in the presence of other factors indicated at the top of the autoradiography picture. The proteins and their positions on the gel are indicated by arrows on the left. The radioactive band resulted from OST1 autophosphorylation (25). (C) ABA-induced growth inhibition in roots. Five-day-old seedlings grown on MS medium were transferred to MS containing 9 μM ABA, and root length was measured 10 d after transfer. Each data point represents the average length ± SE of three experiments with 10 seedlings each. (D) ABA-induced growth arrest in rop11/arac10-1 seedlings. Col-0 and rop11/arac10-1 (SALK_039681) were grown and harvested at the same time. (a–d) Sterilized seeds were plated on MS medium supplemented with 0 μM (a and b) or 2 μM ABA (c and d) at 23 °C for 9 d before photographing. (e and f) Close-up pictures of rop11/arac10-1 (e) and WT (f) show difference in ABA-induced growth arrest. Experiment was conducted five times with similar results. (E) ABA inhibition of germination in the WT and rop11/arac10-1 seeds. Approximately 100 seeds from three independent seed lots of simultaneously grown WT and mutant plants were germinated on different concentrations of ABA. The germination percentage was calculated 2 d after the seeds were plated on MS medium. The experiment was replicated three times. Each value is the average ± SE of three replicates. (F) Stomatal response to ABA. Stomatal apertures were measured with WT and rop11/arac10-1 plants. Data are presented as average value ± SE of three replicates with 10 apertures each. Four independent experiments were conducted with similar results.

Fig. 4.

ABA response of gef mutants and a working model for the FER signaling pathway in the regulation of ABA responses. (A) ABA-induced growth inhibition in roots. The WT, single mutants, gef1/4 double mutant, and gef1/4/10 triple mutant were grown on MS medium for 5 d before transfer to MS containing 12 μM ABA. Root length was then measured 3 d after transfer. Each value is the average ± SE of three replicates of 30 plants. Three separate experiments were conducted. (B) ABA-induced stomatal closure in WT and gef mutants. Leaves were treated with 5 μM ABA for 1 h before stomatal apertures were measured. Each value is the average ± SE of 3 independent experiments with 10 apertures each. (C) Working model for FER pathway in regulating response to ABA. The FER–GEF–ROP/ARAC pathway is an essential module for the auxin response in cell growth. ABI2 is a major component of ABA signaling pathway. The ROP11/ARAC10–ABI2 interaction mediates cross-talk between the auxin and ABA signals. Arrows denote activation, and bars indicate repression. Details are described in the text.