TTL Proteins Scaffold Brassinosteroid Signaling Components at the Plasma Membrane to Optimize Signal Transduction in Arabidopsis

Abstract

Brassinosteroids (BRs) form a group of steroidal hormones essential for plant growth, development, and stress responses. BRs are perceived extracellularly by plasma membrane receptor-like kinases that activate an interconnected signal transduction cascade, leading to the transcriptional regulation of BR-responsive genes. TETRATRICOPEPTIDE THIOREDOXIN-LIKE (TTL) genes are specific for land plants, and their encoded proteins are defined by the presence of protein-protein interaction motives, that is, an intrinsic disordered region at the N terminus, six tetratricopeptide repeat domains, and a C terminus with homology to thioredoxins. TTL proteins thus likely mediate the assembly of multiprotein complexes. Phenotypic, molecular, and genetic analyses show that TTL proteins are positive regulators of BR signaling in Arabidopsis (Arabidopsis thaliana). TTL3 directly interacts with a constitutively active BRASSINOSTEROID INSENSITIVE1 (BRI1) receptor kinase, BRI1-SUPPRESSOR1 phosphatase, and the BRASSINAZOLE RESISTANT1 transcription factor and associates with BR-SIGNALING KINASE1, BRASSINOSTEROID INSENSITIVE2 kinases, but not with BRI1-ASSOCIATED KINASE1. A functional TTL3-green fluorescent protein (GFP) shows dual cytoplasmic plasma membrane localization. Depleting the endogenous BR content reduces plasma membrane localization of TTL3-GFP, while increasing BR content causes its plasma membrane relocalization, where it strengthens the association of BR signaling components. Our results reveal that TTL proteins promote BR responses and suggest that TTL proteins may function as scaffold proteins by bringing together cytoplasmic and plasma membrane BR signaling components.

Figure 1.

TTL3 Interacts with BRI1 In Vivo and In Vitro. (A) Structural model of TTL3 protein predicted in silico using I-TASSER server (Zhang, 2008) and processed by PyMOL (Schrödinger). C, C terminus; N, N terminus. (B) Schematic representation of BRI1 protein and the nine Ser/Thr residues of the JM and CT domains that were substituted by Asp in the BAK1-independent, BRI1-constitutive (phosphomimetic) active form BRI1cyt^JMCT9D (Wang et al., 2008). LRR, leucine rich repeat. Yellow-circled P’s means that BRI1cyt^JMCT9D mimics the phosphorylated form of BRI1. (C) Schematic representations of full-length and different truncated versions of TTL3 protein. Numbers indicate first and last amino acids (a.a.) of TTL3 truncated proteins. Domains and protein fragments interspacing the conserved domains are represented with the same color code as in (A). (D) TTL3ΔN1 interacts with BRI1cyt^JMCT9D in vitro, as shown by a GST-pull-down assay. GST-TTL3ΔN1 and GST-TTL3ΔN3 were detected with anti-GST antibody. MBP-BRI1cyt and MBP-BRI1cyt^JMCT9D were detected using specific anti-BRI1 antibodies (Bojar et al., 2014). Pull-down reflects 20% of the total pulled down proteins. Unbound reflects 1% of the total unbound fraction. (E) BRI1-HA co-IPs with GFP-TTL3 full-length and GFP-TTL3 truncated versions ΔN1, ΔN2, and ΔC1. Numbers indicate first and last amino acids of TTL3 truncated proteins. BRI1-HA was transiently coexpressed in N. benthamiana with GFP-TTL3 full-length and truncated versions, and GFP-tagged protein was immunoprecipitated using anti–GFP-Trap beads. Total (input), IP, and CoIP proteins were analyzed by immunoblotting. Equal loading was confirmed by Coomassie blue staining (CBB) of input samples. GFP- and HA-tagged proteins were detected with anti-GFP and anti-HA antibody, respectively. The amount of coIP BRI1-HA was normalized relative to the amount of GFP-tagged protein from the input, dividing the signal intensity of coIP BRI1-HA by the signal intensity of the each GFP-tagged protein from the input that coIP BRI1-HA.

Figure 2.

TTL1, TTL3, and TTL4 Genes Play a Positive Role in BR Signaling. (A) ttl1, ttl3, ttl4, and ttl134 show root growth hyposensitivity to BR. Statistical analysis of root length measurements of Col-0, ttl, and bak1-4 mutants in control conditions (MS) and in response to eBL. Seedlings were grown in long days for 4 d in half-strength MS agar solidified medium and then transferred to half-strength MS agar solidified medium (MS) or half-strength MS agar solidified medium supplemented with 100 nM eBL (MS + 100 nM eBL), and root length was measure 6 d later. Asterisks indicate statistical differences between mutant versus Col-0 determined by the unpaired t test (*P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001 ****P ≤ 0.0001). Data represent mean values, error bars are sem, n ≥ 35 seedlings per experiment. The experiment was repeated three times with similar results. (B) Root length responses to eBL of wild-type Col-0, ttl134, and BR perception mutants. Seedlings were grown and root length was analyzed as described in (A). Asterisks indicate statistical differences between mutant versus Col-0 as determined by the unpaired t test (***P ≤ 0.001, ****P ≤ 0.0001). Data represent mean values, error bars are sem, n = 30 seedlings per experiment. The experiment was repeated three times with similar results. (C) Defective hypocotyl elongation in ttl mutants. Col-0, ttl3, ttl134, and bak1-4 seedlings were grown for 4 d in long-day photoperiod in half-strength MS agar solidified medium. Seedlings with the same size were then placed in the dark, and hypocotyl elongation was measured 3 d later. Asterisks indicate statistically significant differences between Col-0 and the indicated genotype as determined by the unpaired t test (****P ≤ 0.0001), values are mean, error bars are sem, n = 80 seedlings per experiment. The experiment was repeated twice with similar results. (D) BR-responsive genes DWF4 and CPD show induced expression in ttl134 and bak1-4 relative to Col-0 seedlings. Seeds were germinated in half-strength MS agar solidified medium and grown vertically in long-day photoperiod conditions. Five-day-old seedlings were transferred to half-strength MS liquid medium and after 5 d of acclimation, the relative expression level of DWF4 and CPD was measured by RT-qPCR. The expression of DWF4 and CPD was first normalized to the expression of ACTIN2 and represented relative to the expression of Col-0. The data are shown as mean ± sem from at least three independent biological replicates (pool of 20 seedlings per biological replicate). Asterisks indicate statistically significant differences between the indicated genotype versus Col-0 as determined by the unpaired t test (*P ≤ 0.05, **P ≤ 0.01). The experiment was repeated three times with similar results. (E) Phosphorylation status of BES1 in response to exogenously applied eBL in Arabidopsis Col-0 and ttl134. Ten-day-old seedlings pre-treated for 3 d with the BR biosynthetic inhibitor BRZ to deplete the endogenous pool of BRs were subjected to 10 nM eBL treatment for 0, 30, and 60 min. Total proteins from a pool of 20 seedlings were analyzed by an immunoblot assay with a specific anti-BES1 antibody (Yu et al., 2011). The top band corresponds to pBES1 and the bottom band to dephosphorylated BES1 (BES1). The experiment was repeated two times with similar results. CBB, Coomassie blue.

Figure 3.

ttl134 Mutations Enhance the bri1-301–Defective Phenotype and Partially Reduce the BR Responses in bes1-D. (A) Morphological phenotypes of 5-week-old plants grown in short days. Bar = 1 cm. (B) Detached leaves of 5-week-old plants grown in short days (L1, oldest leaf; L23 youngest leaf). Bar = 1 cm. (C) Root length of 3-d-old seedlings grown in long days in half-strength MS agar solidified medium. Dots represent individual measurements from three independent experiments. n denotes measurement of roots from independent seedlings: Col-0 (n = 92), ttl134 (n = 100), bri1-301 (n = 113), and ttl134 bri1-301 (n = 123). Box plots display the first and third quartiles, split by the median; whiskers extend to include the maximum and minimum values. Different lowercase letters indicate significant differences. Data were analyzed with one-way ANOVA and Tukey´s multiple comparison test; P < 0.05. (D) Hypocotyl length of 7-d-old seedlings grown in long days in half-strength MS agar solidified medium. Dots represent individual measurements from three independent experiments. n denotes measurement of hypocotyls from independent seedlings: Col-0 (n = 44), ttl134 (n = 51), bri1-301 (n = 28), ttl134 bri1-301 (n = 73), bes1-D (n = 27), and ttl134 bes1-D (n = 71). Values are plotted and statistically analyzed as in (C). (E) Root length fold changes of seedlings grown for 7 d in the absence (control) or presence of 500 nM eBL in long days. Values are mean, error bars are sem, and n = 22 seedlings per experiment. Different lowercase letters indicate significant differences. Data were analyzed with one-way ANOVA and Tukey´s multiple comparison test; P < 0.05. The experiment was repeated twice with similar results.

Figure 4.

BRs Regulate the Cytoplasmic/Plasma Membrane Localization of TTL3. (A) to (C) The root growth responses to eBL of the ttl134 triple mutant are complemented in TTL3-GFP 2.4. Seedlings were grown for 4 d in half-strength MS agar solidified medium and then transferred to half-strength MS agar solidified medium (A) or half-strength MS agar solidified medium supplemented with 100 nM brassinolide (B) and root length was measured (C). (A) and (B) Representative photographs of seedlings, 6 d after being transferred to control or eBL treatment. Bar in (A) and (B) = 1 cm. (C) Statistical analysis of root length of Col-0, ttl134, and the complementation line TTL3-GFP 2.4. Asterisks indicate statistically significant differences between the indicated genotype versus Col-0 as determined by the unpaired t test (****P ≤ 0.0001). Data represent mean values, error bars are sem, and n = 30 seedlings per experiment. The experiment was repeated three times with similar results. (D) Expression pattern of TTL3-GFP in 3-d-old TTL3-GFP 2.4 Arabidopsis seedlings. Images were captured using conventional wide field fluorescence microscopy with a GFP filter. Bar = 500 µm. (E) and (F) Longitudinal median section of root tips of a 3-d-old Col-0 (E) and TTL3-GFP 2.4 seedling as observed by laser scanning confocal microscopy (F). Images are a merge of green channel showing TTL3-GFP expression and red channel showing plasma membrane stained with FM4-64. Bar = 20 µm. (G) and (H) Confocal images showing localization of TTL3-GFP in epidermal cells from the root meristematic zone in 4-d-old Arabidopsis TTL3-GFP 2.4 in half-strength MS agar solidified medium, in control conditions (1-h treatment with eBL solvent) (G), or after 1 h of 1 µM eBL treatment (H) in half-strength MS agar liquid medium. Bar = 10 µm (horizontal bar). (I) Quantification of fluorescent protein signal in plasma membrane versus cytoplasm. Line scan measurements spanning membrane and cytoplasm were performed (represented in [G] and [H] as a vertical white line), and representative plot profiles of sample measurements are presented. (J) and (K) Quantification of the cytoplasmic and plasma membrane localization of TTL3-GFP in 4-d-old Arabidopsis TTL3-GFP 2.4 seedlings treated for 1 h with 1 µM eBL (J) or pre-treated for 12 h with 5 µM BRZ prior to 1 µM eBL application for 1 h (K). The number of cells with dual cytoplasmic/plasma membrane localization in meristematic and transition zone was counted for each analyzed root using confocal microscopy. Seedlings were grouped in categories according to the number of cells that presented this dual localization, and the percentage of seedlings displaying each category depicted in the key was calculated. Represented categories in the key indicate the number of cells per seedling with dual cytoplasmic/plasma membrane localization. At least 16 seedlings per treatment, and ∼200 cells (cell from epidermis, cortex, and endodermis all combined) per seedling of the meristematic region of the root tip were analyzed.

Figure 5.

TTL3 Associates with BSK1 and BIN2 and Directly Interacts with BSU1. (A) Yeast-two-hybrid assays to determine the interaction of full-length TTL3, the TTL3 fragment TTL3ΔN1 (amino acids 204 to 691), and the TTL3 fragment TTL3ΔN2 (amino acids 371 to 691) with BIN2 and BSU1. Growth on plasmid-selective media (left column) and interaction-selective media (lacking adenine [-ade], right column) are shown. (B) BSK1 co-immunoprecipitates with TTL3. BSK1-HA, and GFP-TTL3 were transiently expressed in N. benthamiana. GFP-TTL3 was IP with anti-GFP Trap beads. Total (input), IP, and CoIP proteins were analyzed by immunoblotting. Equal loading was confirmed by Coomassie blue staining (CBB) of input samples. GFP-TTL3 and BSK1-HA were detected with anti-GFP and anti-HA antibody, respectively. (C) BSU1 co-immunoprecipitates with TTL3. GFP-TTL3 and BSU-HA proteins were transiently expressed in N. benthamiana, IP, and analyzed as described in (B). GFP-TTL3 and BSU1-HA were detected with anti-GFP and anti-HA antibodies, respectively. (D) BIN2 co-immunoprecipitates with TTL3. BIN2-HA and GFP-TTL3 proteins were expressed in N. benthamiana, IP, and analyzed as described in (B). GFP-TTL3 and BSU1-HA were detected with anti-GFP and anti-HA, respectively. (E) TTL3 promotes BIN2 depletion. BIN2-HA with and without GFP-TTL3 was expressed in N. benthamiana. Protein extracts were analyzed by immunoblotting. Equal loading was confirmed by Coomassie blue staining (CBB) of input samples. GFP-TTL3 and BIN2-HA were detected with anti-GFP and anti-HA antibody, respectively. Bottom graph represents the signal density of BIN2-HA coexpressed with or without GFP-TTL3 in N. benthamiana was quantified based on the six biological repeats. The immunoblot signal intensity of BIN2-HA coexpressed with GFP-TTL3 was normalized to the immunoblot signal intensity of BIN2-HA coexpressed with an empty vector. Asterisks indicate statistical differences as determined by the unpaired t test (***P ≤ 0.001).

Figure 6.

TTL3 Interacts with BZR1 and Regulates Its Cytoplasmic/Nuclear Localization. (A) Yeast-two-hybrid assays to determine the interaction of BZR1 with TTL3, the TTL3 fragment TTL3ΔN1 (amino acids 204 to 691), the TTL3 fragment TTL3ΔN2 (amino acids 371 to 691), and BIN2. Interaction of BZR1 with a fragment of Simian virus 40 large T-antigen (AD-AgT) was also included to show BD-BZR1 self-activation capacity. Growth on plasmid-selective media (left column) and interaction-selective media (lacking adenine, -ade], right column) are shown. (B) TTL3 co-immunoprecipitates with BZR1. TTL3-HA and BZR1-GFP were transiently expressed in N. benthamiana. BZR1-GFP was IP with anti-GFP Trap beads. Total (input), IP, and CoIP proteins were analyzed by immunoblotting. Equal loading was confirmed by Coomassie blue staining (CBB) of input samples. BZR1-GFP and TTL3-HA were detected with anti-GFP and anti-HA, respectively. The top band corresponds to phosphorylated BZR1 (pBZR1-GFP) and the bottom band to dephosphorylated BZR1 (BZR1-GFP). (C) Co-IP of BZR1-HA with TTL3-GFP expressed in transfected Arabidopsis Col-0 protoplasts. Samples were analyzed as in (B). Protoplasts cotransfected with free GFP and BRI1-HA, were used as a negative control for Co-IP. Equal loading was confirmed by Ponceau staining of input samples. TTL3-GFP and free GFP were detected with anti-GFP antibody and BRI1-HA was detected with anti-HA antibody. Asterisk indicates GFP that results from proteolytic cleavage of TTL3-GFP. Red arrow indicates an artifact from imaging the blot with high sensitivity using an Azure c300 Chemiluminescent Western Blot Imaging System. (D) TTL3 abolishes the cytoplasmic retention of BZR1 by BIN2. Subcellular localization of BZR1-GFP alone, coexpressed with BIN2-HA, and with BIN2-HA and TTL3-HA in N. benthamiana leaves. Images of the GFP signal were obtained using laser scanning confocal microscopy. Images show a single equatorial plane in N. benthamiana leaves. Bar = 20 μm. The experiment was repeated three times with similar results. (E) Immunoblot analysis of the BZR1-GFP proteins transiently expressed alone, coexpressed with BIN2-HA, and coexpressed with BIN2-HA and TTL3-HA in N. benthamiana leaves observed by confocal microscopy in (D). Proteins were analyzed by immunoblotting. Equal loading was confirmed by Coomassie blue staining (CBB) of input samples. BZR1-GFP was detected with anti-GFP antibody, while TTL3-HA and BIN2-HA were detected with anti-HA antibody. In the anti-GFP blot, the top band corresponds to phosphorylated BZR1 (pBZR1-GFP) and the bottom band to dephosphorylated BZR1 (BZR1-GFP).

Figure 7.

Coexpression of TTL3 Enhances pBZR1-BSK1 Interaction. (A) BiFC shows strong association of BSK1 with BRI1, BSU1, and BIN2 and weak association with BZR1. N. benthamiana leaves were co-agroinfiltrated with the Agrobacterium strains harboring a construct to express the BSK1 protein fused to the N-terminal half of YFP and the BRI1, BSU1, BIN2, or BZR1 proteins fused to the C-terminal half of YFP and observed under a laser scanning confocal microscope. Strong fluorescence signals are observed when BSK1-nYFP is coexpressed with BRI1-cYFP, BSU1-cYFP, or BIN2-cYFP. A faint YFP signal is observed when BSK1-nYFP is coexpressed with BZR1-cYFP. From left to right columns, images show BiFC YFP fluorescence in green and bright-field. Bar = 20 µm. The experiment was repeated two times with similar results. (B) Expression of TTL3 increases the weak BiFC association of BSK1 and BZR1. N. benthamiana leaves were co-agroinfiltrated with the Agrobacterium strains harboring the corresponding constructs to express the BSK1 protein fused to the N-terminal half of YFP and the BZR1 protein fused to the C-terminal half of YFP. N. benthamiana leaves were pre-treated with 5 µM eBL for 3 h before confocal imaging analysis. Coexpression of TTL3-HA together with BSK1-nYFP and BZR1-cYFP highly enhances the GFP signal. From left to right columns, BiFC YFP fluorescence in green and bright field. Bar = 20 µm. The experiment was repeated three times with similar results. (C) Quantification of the BiFC fluorescence intensity of BSK1 and BZR1 in the presence or absence of TTL3-HA described in (B). Asterisks indicate statistical differences between BiFC fluorescence intensity of BSK1 and BZR1 in the absence or presence of TTL3-HA determined by the unpaired t test (***P ≤ 0.001). Data represent mean values, error bars are sem, and n = 5 randomly chosen regions of infiltrated leaves. The experiment was repeated three times with similar results. a.u., arbitrary units. (D) Immunoblot analysis reveals similar amounts of BSK1-nYFP and BZR1-cYFP when coexpressed with or without TTL3-HA. Proteins were transiently expressed as described in (B). Equal loading was confirmed by Coomassie blue staining (CBB) of total proteins. BSK1-nYFP contains a myc tag (BSK1-myc-nYFP) and was detected using anti-myc antibody, while BZR1-cYFP contains a HA tag (BZR1-HA-cYFP) and was detected using an anti-HA antibody. TTL3-HA was also detected with an anti-HA antibody. The experiment was repeated three times with similar results.