EXO70D isoforms mediate selective autophagic degradation of type-A ARR proteins to regulate cytokinin sensitivity

Abstract

The phytohormone cytokinin influences many aspects of plant growth and development, several of which also involve the cellular process of autophagy, including leaf senescence, nutrient remobilization, and developmental transitions. The Arabidopsis type-A response regulators (type-A ARR) are negative regulators of cytokinin signaling that are transcriptionally induced in response to cytokinin. Here, we describe a mechanistic link between cytokinin signaling and autophagy, demonstrating that plants modulate cytokinin sensitivity through autophagic regulation of type-A ARR proteins. Type-A ARR proteins were degraded by autophagy in an AUTOPHAGY-RELATED (ATG)5-dependent manner, and this degradation is promoted by phosphorylation on a conserved aspartate in the receiver domain of the type-A ARRs. EXO70D family members interacted with type-A ARR proteins, likely in a phosphorylation-dependent manner, and recruited them to autophagosomes via interaction of the EXO70D AIM with the core autophagy protein, ATG8. Consistently, loss-of-function exo70D1,2,3 mutants exhibited compromised targeting of type-A ARRs to autophagic vesicles, have elevated levels of type-A ARR proteins, and are hyposensitive to cytokinin. Disruption of both type-A ARRs and EXO70D1,2,3 compromised survival in carbon-deficient conditions, suggesting interaction between autophagy and cytokinin responsiveness in response to stress. These results indicate that the EXO70D proteins act as selective autophagy receptors to target type-A ARR cargos for autophagic degradation, demonstrating modulation of cytokinin signaling by selective autophagy.

Keywords: carbon starvation; concanamycin A; cytokinin signaling; selective autophagy.

Fig. 1.

ARR4 is degraded by the autophagy pathway in Arabidopsis roots. (A) Response of root-expressed ARR4 to ConcA. Seedlings carrying pUBQ10::ARR4:GFP constructs were treated with ConcA and transferred to dark to induce carbon starvation. ARR4 protein quantities in the roots analyzed by immunoblot assays using anti-GFP (α-GFP) antibodies. Anti-tubulin (α-tub) served as loading control. Rel. quantities represent ratio of intensity of α-GFP to α-tub band relative to ratio at 0 h. (B) Disruption of autophagosome-forming ATG5 gene elevated the levels of ectopically expressed ARR4 proteins. The progeny of a cross between pUBQ10::ARR4:GFP-expressing plants described in A and atg5-1 and wild-type plants were grown under normal conditions for 9 d and for additional 1 d under carbon starvation conditions. Seedlings were either untreated (C-0) or treated with DMSO mock control (C-1) and ConcA (ConcA) as described above, prior to immunoblot assay. Rel. quantities represent ratio of intensity of α-GFP to α-tub band relative to ratio of WT band. For immunoblot assay of ARR4:GFP;atg5-1, Rel. quantities represent intensity ratio relative to bands of C-0. (C) Representative confocal micrograph of root of 5-d-old seedlings described in B, treated with ConcA or DMSO as mock control and exposed to carbon starvation for 18 h prior to imaging. (Right) Quantification of the number of GFP-containing vacuolar puncta. Data represent the average number of puncta per 100 µm², n = 23, analyzed with one-way ANOVA followed by Tukey–Kramer multiple comparison analyses; P < 0.05. Different letters represent statistically different means. (D) Representative confocal micrograph of root of T1 seedlings of wild-type Col-0 transformed with pARR4::ARR4:GFP construct. Seedlings were treated as described in C. (Right) Graph represents average number of puncta per 5,000-μm² area (n = 60, from 20 independent T1 seedlings). (E) Intracellular colocalization of RFP signal resulting from ARR4:RFP with the GFP signal from GFP:ATG8f. Seedling coexpressing both constructs were treated with ConcA as described in C. (Scale bar, 10 µm.) The magnified area enclosed by the yellow box indicates section of vacuole showing colocalization of ARR4:RFP- and GFP:ATG8f-containing vesicles. Graph represents mean (n = 23) number of RFP- and GFP-containing puncta per 50 µm² of vacuole area. (For C, D, E: Scale bar, 10 μm.) For D, E, ****statistical differences at P < 0.0001 using Student’s t test.

Fig. 2.

Interaction between EXO70Ds and type-A ARRs results in the destabilization of the type-A ARRs. (A) Yeast two-hybrid (Y2H) assay showing pairwise interactions between members of the EXO70D subclade and representative type-A ARRs. The EXO70D paralogs were cloned into the Gal4 DNA activation domain prey vector while the type-A ARRs (WT) with their respective phosphor-dead (A) and phosphor-mimic (E) mutations were cloned as baits in Gal4 DNA binding domain vectors. Interactions were accessed on -Leu-Trp-His-Ura (-L -W -H -Ura) quadruple dropout media. The -Leu-Trp (-L -W) served as control media. Interactions between the type-A ARRs and Arabidopsis Histidine Phosphotransfer Protein 2 (AHP2) served as positive control. (B) Y2H assay of interactions of N-terminal (EXO70D3^N-term) or C-terminal (EXO70D3^C-term) domains of EXO70D3 with the representative type-A ARRs described in A. See SI Appendix, Fig. S3A for details on N-term and C-term. Interaction assays are similar to A. (C) EXO70D3^C-term interacts with phosphor-mimic mutants of ARR5 (ARR5^D87E) in coimmunoprecipitation assay. Leaves of N. benthamiana were infiltrated with plasmids expressing myc:EXO70D3^C-term or HA:ARR5^D87E. The input extracts and the myc-immunoprecipitated proteins (IP) were analyzed by immunoblotting with anti-HA and anti-myc. (D) Ectopically expressed ARR4:GFP accumulates in roots tips of exo70D1,2,3 triple loss-of-function mutant. Confocal microscopy images indicating the expression of pUBQ10::ARR4:GFP in wild-type (Top) and exo70D1,2,3 mutant (Bottom) plants. (Scale bars, 10 µm.) (Right) Images are quantified by measuring signal intensity of individual nuclei, after background normalization. The graph represents the average signal measurement from 22 images of each genotype. Data were analyzed by unpaired Student’s t test. ****Statistical difference at P < 0.001 (4.025e-06). (E) Anti-GFP immunoblot analyses of roots of 10-d-old Arabidopsis seedlings, transgenic seedlings expressing pUBQ10::ARR4:GFP in wild type (WT) or exo70D1,2,3 (mut) described in D. Rel. quantities represent ratio of intensity of α-GFP to α-tub band relative to ratio of bands from WT (ARR4:GFP).

Fig. 3.

EXO70Ds mediate cytokinin responses by recruiting type-A ARRs to the ATG8-tagged autophagy machinery for degradation. (A) Representative confocal micrographs of the root elongation zones of ectopically expressed ARR4:GFP in wild type (WT) and exo70D1,2,3 mutants incubated with ConcA or DMSO (as control). Quantification of the number of GFP-containing puncta is as described in Fig. 1C. Different letters represent values that are statistically different (n = 23) at the indicated P value. (B) Disruption of EXO70D genes inhibits autophagy-mediated destabilization of type-A ARR proteins. Seedlings were treated with ConcA followed by immunoblot assays using anti-GFP. α-tub was a loading control. Rel. quantities represent the ratio of intensity of α-GFP to α-tub band relative to ratio of bands in WT (ARR4:GFP) at 0 h. (C) Yeast two-hybrid (Y2H) interaction of ATG8 paralogs with N-terminal (EXO70D3^N-term) or C-terminal (EXO70D3^C-term) domains of EXO70D3. (D) Y2H interaction of prey ATG8 paralogs with N-terminal domains of WT (WT-EXO70D3^N-term) or W234A/V237A AIM-mutated (mut-EXO70D3^N-term) EXO70D3 as bait. (E) Functional AIM is required for the EXO70D3-dependent destabilization of ARR4 proteins. N. benthamiana leaves were transiently cotransformed with fixed amount of HA:ARR4 and increasing titer of myc-tagged full-length WT (WT-EXO70D3) or W234A/V237A mutant (mut-EXO70D3) EXO70D3. α-GFP served as transfection control. Rel. quantities represent the ratio of band intensities of α-HA:α-myc:α-GFP (α-HA/α-myc/α-GFP) relative to the ratio of lane 4 which was set at 1.0. (F) Representative confocal microscopy image of root of Arabidopsis pUBQ10::EXO70D3:GFP seedlings treated with ConcA or DMSO (as control). Quantification of the number of EXO70D3:GFP-containing puncta. Data represent mean of 24 independent images. ****Values that are statistically different at P < 5.066 × 10⁻⁰³²¹. (G) Colocalization of EXO70D3:GFP and mCherry:ATG8e in roots of Arabidopsis seedlings carrying plasmids expressing both genes. (H) Colocalization of mCherry:EXO70D3 and ARR4:GFP in elongation zone of 5-d-old Arabidopsis seedlings ectopically expressing both genes. For G and H, seedling treatment and imaging is as described in Fig. 1E. The magnified areas enclosed by the yellow boxes show colocalization of vesicles containing mCherry:ATG8e and EXO70D3:GFP, and Cherry:EXO70D3 and ARR4:GFP, respectively. (I) Root elongation assay showing the cytokinin response of exo70D1,2,3 and WT seedling roots. Seedlings were grown on MS media supplemented with BA (cytokinin) or NaOH (control). (Scale bar, 1 cm.) Different letters represent statistically different means (n ≥ 13) at P < 0.05. (J) The autophagic flux of ARR4 is dependent on the phosphorylation status of the conserved aspartate. Representative confocal micrograph of root of seedlings expressing the indicated GFP-tagged proteins, treated with ConcA (Upper) or DMSO (Lower). Graph represents average number of puncta per 5,000-μm² area (n = 48, from 8 independent transformants). *** and **** represent statistically different values at P = 0.0001 and P < 0.0001, respectively. (K) Cytokinin enhances the autophagic flux of ARR4. Representative confocal micrograph of roots of seedlings treated with ConcA (+ConcA) (Upper) or DMSO (-ConcA) (Lower) under carbon starvation conditions for 10 h. Seedlings were then treated with 5 µM BA (+BA) (Left) or NaOH as vehicle control (-BA) (Right) for 4 h prior to imaging. Graph represents average number of puncta per 66,000-pixel² area (for n ≥ 30). ****Values that are statistically different between treatments. Note that the BA × ConcA interaction was also significant. (Scale bar in A, F, G, H, J, K, 10 μm.)

Fig. 4.

EXO70Ds and type-A ARRs modulate plant response to fixed-carbon starvation. Seedlings were grown for 6 wk on potted soil under short-day conditions of 16/8 h day/night at 22 °C. Pots were transferred to dark for 7, 9, 11, and 13 d and recovered in the light for 7 d. (A) Representative images of exo70D1,2,3 triple loss-of-function mutants and arr5-1 and some high-order type-A ARR loss-of-function mutants (arr3,4,5,6; arr3,4,8,9; arr5,6,8,9; arr3,4,5,6,7,8,9,15) following 13-d-dark treatment. Col-0 and atg5-1 served as wild-type and autophagy deficient controls, respectively. (B and C) Quantification of survival of exo70D1,2,3 (B) and type-A ARR mutants (C) in response to carbon starvation. Survival was estimated as the percentage of plants with new leaves after the dark treatment. Values represent mean ± SEM percentage survival of three biological replicates. Each biological replicate consisted of eight plants per genotype per treatment.

Fig. 5.

Model of autophagic and proteasome regulation of type-A ARRs. In the presence of cytokinin, type-A ARRs are phosphorylated on the conserved aspartate in the receiver domain. Phosphorylated type-A ARRs are stabilized and negatively regulate cytokinin responses. To regulate this constitutive type-A ARR action, EXO70Ds recruit the phosphorylated type-A ARR to the autophagosome by interacting with ATG8 isoforms. This EXO70D-mediated autophagic mechanism presents a more rapid degradation pathway for type-A ARRs. In the absence of cytokinin, unphosphorylated type-A ARRs are ubiquitinated and shuttled to the 26S proteasome for degradation. This 26S proteasome pathway is probably activated for a prolong degradation of type-A ARRs. Black arrow: phosphate transfer; green line: protein–protein interaction; purple arrow: protein degradation; red line: negative feedback regulation.