Arabidopsis ALIX Regulates Stomatal Aperture and Turnover of Abscisic Acid Receptors

Abstract

The plant endosomal trafficking pathway controls the abundance of membrane-associated soluble proteins, as shown for abscisic acid (ABA) receptors of the PYRABACTIN RESISTANCE1/PYR1-LIKE/REGULATORY COMPONENTS OF ABA RECEPTORS (PYR/PYL/RCAR) family. ABA receptor targeting for vacuolar degradation occurs through the late endosome route and depends on FYVE DOMAIN PROTEIN REQUIRED FOR ENDOSOMAL SORTING1 (FYVE1) and VACUOLAR PROTEIN SORTING23A (VPS23A), components of the ENDOSOMAL SORTING COMPLEX REQUIRED FOR TRANSPORT-I (ESCRT-I) complexes. FYVE1 and VPS23A interact with ALG-2 INTERACTING PROTEIN-X (ALIX), an ESCRT-III-associated protein, although the functional relevance of such interactions and their consequences in cargo sorting are unknown. In this study we show that Arabidopsis (Arabidopsis thaliana) ALIX directly binds to ABA receptors in late endosomes, promoting their degradation. Impaired ALIX function leads to altered endosomal localization and increased accumulation of ABA receptors. In line with this activity, partial loss-of-function alix-1 mutants display ABA hypersensitivity during growth and stomatal closure, unveiling a role for the ESCRT machinery in the control of water loss through stomata. ABA-hypersensitive responses are suppressed in alix-1 plants impaired in PYR/PYL/RCAR activity, in accordance with ALIX affecting ABA responses primarily by controlling ABA receptor stability. ALIX-1 mutant protein displays reduced interaction with VPS23A and ABA receptors, providing a molecular basis for ABA hypersensitivity in alix-1 mutants. Our findings unveil a negative feedback mechanism triggered by ABA that acts via ALIX to control the accumulation of specific PYR/PYL/RCAR receptors.

Figure 1.

ALIX Interaction with PYL ABA Receptors. (A) The structure of ALIX showing the Bro1 domain (Bro1; amino acids 1 to 413) and the coiled coils plus the proline-rich region (∆Bro1; amino acids 405 to 846) are shown in the top panel. Y2H assays showing interaction of PYL ABA receptors with either Bro1 or ∆Bro1 domains. Transformed yeast cells were grown in synthetic drop-out (SD) medium lacking tryptophan and leucine (-WL) as a transformation control and in SD-WL media lacking adenine (–WLA), histidine (–WLH), or both (–WLHA) for interaction assays. Cotransformation with empty plasmids was used as negative control. (B) Bacteria-purified PYL proteins fused to a 6x His tag (His-PYL) were pulled down on incubation with recombinant MBP-ALIX or MBP bound to amylose resin. Anti-His was used in immunoblots (IBs) to detect His-PYL fusions, and anti-MBP for MBP-ALIX and MBP. (C) HA-PYL4 was pulled down on incubation of oeHA-PYL4 plant extracts with recombinant MBP-ALIX or MBP bound to amylose resin. Protein extracts from wild-type (Col-0) plants were used as negative control. Anti-HA was used to detect HA-PYL4 and anti-MBP for MBP-ALIX and MBP. (D) In vivo interaction of ALIX with PYL4 assessed by BiFC. Confocal images were taken of N. benthamiana leaf epidermal cells expressing different BiFC construct combinations together with mCherry-ARA7, a MVB marker. Reconstitution of YFP fluorescence shows that ALIX and PYL4 constructs directly interact in mCherry-ARA7-labeled MVBs (i.e., punctae indicated by arrows in Overlay image). Bright-field (BF) images of the leaf areas are shown. Bars = 15 and 20 µm. The numbers of intracellular punctae per leaf section agroinfiltrated (y axis; dots per 50 × 50 µm field) as well as the percentage of colocalization of YFP-fluorescent and mCherry-ARA7 labeled vesicles (y axis; percentage of colocalization) with each construct combination (x axes) are quantified below the micrographs. Results were obtained from 10 fields from five biological replicates (two fields/replicate).

Figure 2.

alix-1 Mutants Show Enhanced Sensitivity to ABA. (A) Representative photographs of 12-d-old wild-type (Col-0), complemented line GFP-ALIX/alix-1 (labeled as GFP-ALIX), oeHA:PYL4 (HA-PYL4), alix-1, ABA insensitive cra1 and ABA hypersensitive hab1-1 abi1-2 mutants grown in the presence of 0.5 µM ABA. (B) Percentage of seeds germinated (radicle emergence) in the presence of 1 µM ABA at 3 d after sowing. Genotypes analyzed were as in (A). (C) ABA-mediated inhibition of seedling establishment (emergence of the first true leaves) of the same genotypes analyzed in (A) that were grown in medium lacking or supplemented with 0.5 µM ABA. (D) Root length measurements of seedlings germinated in the presence or absence of 0.5 µM ABA after 12 d, or in the presence of 1 µM ABA after 15 d. Genotypes were as in (A). (E) ABA concentration (pmol/g) in shoot and roots of 14-d-old seedlings for wild-type (Col-0), GFP-ALIX and alix-1 mutant. hab1-1 abi1-2 mutants were used as the ABA hypersensitive control. (F) Photographs of representative plants analyzed in (D). *p < 0.05; **p < 0.01 (Student’s t test; Supplemental Data Set 2) with respect to the wild type in the same experimental conditions. In all cases, data are means of three biological replicates (n = 20 in each replicate). Error bars represent sd. MS medium was used as a control in all assays.

Figure 3.

Reduced Water Loss and Stomatal Aperture in alix-1 Mutants. (A) Kinetics of the loss of fresh weight in 15-day-old seedlings of wild-type (Col-0), complemented line GFP-ALIX/alix-1 (labeled as GFP-ALIX), oeHA-PYL4 (HA-PYL4) and alix-1 genotypes. The cra1 and hab1-1 abi1-2 mutants were used as ABA insensitive and hypersensitive controls, respectively. Plants were exposed for 40 min to the drying environment of a laminar flow hood. Values shown are averages ±se from three replicates (n = 15 in each replicate). (B) IR imaging was used to analyze foliar temperature in wild-type (Col-0), complemented line GFP-ALIX/alix-1, and alix-1 plants. The ost2-2 mutant, which displays incompletely closed stomata, was used as a control. Correspondence between false colors and temperatures (degrees Celsius) in IR images is shown. (C) Kinetics of stomatal aperture in response to light in leaves of wild-type (Col-0), GFP-ALIX/alix-1, and alix-1 plants. Values shown are averages ±se from three replicates (n = 70 in each replicate). (D) Confocal imaging was used to visualize vacuolar morphology in guard cells of wild-type (WT, Col-0) and alix-1 plants expressing the tonoplast marker YFP-VAMP711. Plants were grown under short days for 21 d before leaves were collected. Bars = 10 and 5 µm, respectively. (E) Stomatal aperture measurements in response to FC in dark- and light-treated leaves of wild-type (Col-0), GFP-ALIX/alix-1, and alix-1 plants. Values shown are averages ±se from three replicates (n = 70 in each replicate). *p < 0.05; **p < 0.01 (Student’s t test) with respect to the wild type in the same experimental conditions.

Figure 4.

Hypersensitivity to ABA in alix-1 Stomata. (A) Confocal fluorescence images showing GFP-ALIX localization in the cytoplasm and punctuate structures of wild-type guard cells. Bar=10 µm. (B) Analysis of stomatal closure in response to different concentrations of ABA in alix-1 mutant, wild-type (Col-0), and GFP-ALIX/alix-1 (labeled as GFP-ALIX) lines. Data are presented as percentage relative to each genotype under light conditions (100% stomatal aperture). Values shown are averages ±se from three replicates (n = 70 in each replicate). (C) RT-qPCR-analysis of the expression of ABA responsive genes ABI1 and MYB60 in soil-grown alix-1 mutants, wild-type (Col-0), and GFP-ALIX/alix-1 plants. **p < 0.01 (Student’s t test; Supplemental Data Set 2) with respect to the wild type in the same experimental conditions. Data are means of three biological replicates with two technical replicates per sample. Error bars represent sd.

Figure 5.

Impaired Trafficking and Vacuolar Degradation of ABA Receptors in alix-1 Mutants. (A) Confocal images of wild-type (Col-0) and alix-1 mutant root cells expressing GFP-PYL4 on treatment or not with wortmannin (WM) and stained with LysoTracker Red. Bars = 10 and 25 µm. Arrows indicate representative WM-enlarged vesicles. (B) Immunoblot (IB) showing accumulation of endogenous PYL4 in wild-type (Col-0) and alix-1 seedlings grown in the presence or absence of 0.3 µM ABAμ. Anti-RPT5 was used as loading control. (C) Immunoblot (IB) analyses of PYL4 levels in 8-d-old wild-type (Col-0) and alix-1 seedlings treated with 50 µM CHX for 8 h. Anti-RPT5 was used as loading control. (D) Immunoblot (IB) analysis to track the vacuolar delivery of GFP-PYL4 in the wild-type (WT, Col-0) and alix-1 backgrounds. Anti-GFP was used in immunoblots to detect GFP-PYL4 and the GFP-core signal. Band intensity was quantified using Fiji (ImageJ 1.52i). Ponceau staining was used as loading control as indicated. (E) Isolation of microsomes from postnuclear fractions (Input) of 8-d-old wild-type (Col-0) or alix-1 seedlings treated or not with ABA for 3 h. Endogenous anti-PYL4 was used to evaluate PYL4 abundance in the cytosolic and microsomal (Memb.) fractions. Ponceau staining was used as loading control as indicated. (F) Immunoblot (IB) analysis of PYL4 levels in nuclear protein extracts from 8-d-old wild-type (Col-0) or alix-1 seedlings treated or not with ABA for 3 h. Anti-RPT5 was used as loading control. (G) Immunoblot to test the specificity of anti-PYL4. Protein extracts from 8-d-old wild-type (Col-0), single mutants pyr1 and pyl4 and quadruple mutant pyr1 pyl4 pyl5 pyl8 were used. Arrows indicate the position of endogenous PYR1 and PYL4 proteins in the immunoblot. Anti-RPT5 was used as loading control.

Figure 6.

Reduced PYL Function in pent alix-1 Plants Suppresses ABA Defects Caused by alix-1 Mutation. (A) Representative photographs of 15-d-old wild-type (Col-0), alix-1, complemented line gALIX/alix-1, and pentuple pyr1 pyl1 pyl4 pyl5 pyl8 in wild-type (pent) or alix-1 (pent alix-1) backgrounds grown in the presence of 1 µM ABA. (B) ABA-mediated inhibition of seedling establishment (emergence of the first true leaves) of the same genotypes analyzed in (A) that were grown in medium lacking or supplemented with 1 µM ABA. Data are means of three biological replicates (n = 20 in each replicate). Error bars represent sd. MS medium was used as a control in all assays. (C) Kinetics of the loss of fresh weight in 15-d-old seedlings of the genotypes used in (A). Plants were exposed for 60 min to the drying environment of a laminar flow hood. Values shown are averages ±se from three replicates (n = 15 in each replicate). (D) Analysis of foliar temperature in wild-type (Col-0), alix-1, pent and pent alix-1 plants using IRimaging. Correspondence between false colors and temperatures (°C) in IR images is shown. (E) Analysis of stomatal closure in response to different concentrations of ABA in the same genotypes analyzed in (A). Values shown are averages ±se from three replicates (n = 100 in each replicate). In (B) and (E) *p < 0.05; **p < 0.01 (Student’s t test; Supplemental Data Set 2) with respect to the wild type in the same experimental conditions.

Figure 7.

ALIX-1 Mutant Protein Displays Reduced Interaction with ABA Receptors and the ESCRT Component VPS23A. (A) Y2H assays for the interaction between the Bro1 domain of ALIX or a variant containing the alix-1 mutation (Bro1mut) and PYL ABA receptors. Transformed yeast cells were grown in synthetic drop-out medium lacking tryptophan and leucine (SD-WL) as a transformation control and in SD-WL media lacking adenine (−WLA), histidine (−WLH), or both (−WLHA) for interaction assays. 3-amino-1,2,4-triazole (3AT; 2mM) was added to better visualize positive interactions. Empty vectors were used as negative controls. (B) to (D) BiFC assays showing impaired interaction of an ALIX version containing the alix-1 mutation (ALIX-1) with PYL4 (B), VPS23A (C), and wild-type ALIX (D). Bars = 30 µm (A) and 40 µm (C and D). Colocalization of YFP fluorescence by ALIX-PYL4 interaction with mCherry-ARA7-labeled MVBs is indicated by arrows in the Overlay image in (B). Bright-field (BF) images of the corresponding leaf areas are also shown in (B) to (D). The numbers of intracellular punctae per leaf section (y axis; dots per 50 × 50 µm field) agroinfiltrated with each construct combination (x axes) are quantified. Results were obtained from 10 fields from five biological replicates (2 fields/replicate).

Figure 8.

Proposed Model for the Role of ALIX in the Endosomal Trafficking of ABA Receptors. Soluble PYL4 proteins transiently bind to the plasma membrane via interaction with calcium binding CAR proteins undergoing subsequent endosomal trafficking for vacuolar degradation. MVB sorting of ABA receptors is mediated by the ESCRT machinery aided by ALIX. Vacuolar proteolysis of PYL4 is enhanced in the presence of ABA, likely as a desensitizing mechanism to this hormone. alix-1 mutation impairs the interaction with PYL4 and the ESCRT-I component VPS23A, hampering the internalization of PYL4 into intraluminal vesicles in MVBs. Thus, alix-1 leads to the accumulation of cytosolic and membrane-associated PYL4 that, together with increased ABA content in this mutant, increases sensitivity to ABA. Exacerbated response to ABA, together with vacuolar defects inherent to the alix-1 mutation, causes higher stomatal closure compared with wild-type plants.