SMAX1-LIKE7 Signals from the Nucleus to Regulate Shoot Development in Arabidopsis via Partially EAR Motif-Independent Mechanisms

Abstract

Strigolactones (SLs) are hormonal signals that regulate multiple aspects of shoot architecture, including shoot branching. Like many plant hormonal signaling systems, SLs act by promoting ubiquitination of target proteins and their subsequent proteasome-mediated degradation. Recently, SMXL6, SMXL7, and SMXL8, members of the SMAX1-LIKE (SMXL) family of chaperonin-like proteins, have been identified as proteolytic targets of SL signaling in Arabidopsis thaliana However, the mechanisms by which these proteins regulate downstream events remain largely unclear. Here, we show that SMXL7 functions in the nucleus, as does the SL receptor, DWARF14 (D14). We show that nucleus-localized D14 can physically interact with both SMXL7 and the MAX2 F-box protein in a SL-dependent manner and that disruption of specific conserved domains in SMXL7 affects its localization, SL-induced degradation, and activity. By expressing and overexpressing these SMXL7 protein variants, we show that shoot tissues are broadly sensitive to SMXL7 activity, but degradation normally buffers the effect of increasing SMXL7 expression. SMXL7 contains a well-conserved EAR (ETHYLENE-RESPONSE FACTOR Amphiphilic Repression) motif, which contributes to, but is not essential for, SMXL7 functionality. Intriguingly, different developmental processes show differential sensitivity to the loss of the EAR motif, raising the possibility that there may be several distinct mechanisms at play downstream of SMXL7.

Figure 1.

SL Signaling Components Function in the Nucleus. (A) to (C) Subcellular localization of SMXL7-YFP (A), D14-CFP (B), and MAX2-CFP (C) in N. benthamiana epidermal cells, transiently expressed from the 35S promoter. (D) Primary rosette branching levels in Col-0, max2-1, and smxl6-4 smxl7-3 smxl8-1 max2-1 transformed with nothing (3rd bar), SMXL7pro:SMXL7-VENUS (4th bar), 35Spro:SMXL-YFP (two independent homozygous lines, 5th and 6th bars), and 35Spro:SMXL7^ΔNLS-YFP, in which the nuclear localization signal has been deleted (two independent homozygous lines, 7th and 8th bars). Plants were grown in long days for 7 weeks. n = 13 to 22; error bars indicate se; bars with the same letter are not significantly different from one another (ANOVA + Tukey HSD, P < 0.05). (E) to (G) Subcellular localization of SMXL7-YFP (E), D14-CERULEAN (F), and MAX2-GFP in Arabidopsis root meristems, in homozygous transgenic lines expressing the fusion proteins from the 35S promoter ([E] and [F]) or MAX2 promoter (G). (H) and (I) Expression of SMXL7^d53-VENUS (in which the protein has been stabilized by replacing amino acids 812RGKTVV817 with T) (H) and D14-CERULEAN (I) in transverse hand sections through Arabidopsis inflorescence stems, in homozygous transgenic lines expressing the fusion proteins from their native promoters. (J) Subcellular localization in an Arabidopsis root meristem of SMXL7^ΔNLS-YFP in homozygous transgenic lines expressing the fusion protein from the 35S promoter. (K) Rosette branching levels in Col-0 and d14-1 transformed with nothing (2nd bar) and D14pro:D14-CERULEAN (three independent lines, 3rd to 5th bars), D14pro:D14^NLS-VENUS in which a strong nuclear localization signal has been added (three independent lines, 6th to 8th bars), and D14pro:D14^palm/myr-CERULEAN in which a palmitoylation/myristoylation motif has been added (three independent lines, 9th to 11th bars). The data are taken from a larger data set and show the most, median, and least rescued lines for each construct. The full data set is shown in Supplemental Figure 1. Plants were grown in short days for 4 weeks, grown in long days until the inflorescence stem was 10 cm long, and then decapitated. Primary rosette branches were counted 10 d later (Greb et al., 2003). n = 13 to 20; error bars indicate se; bars with the same letter are not significantly different from one another (ANOVA + Tukey HSD, P < 0.05). (L) to (N) Subcellular localization of D14-CERULEAN (L), D14^NLS-VENUS (M), and D14^palm/myr-CERULEAN (N) in N. benthamiana epidermal cells, transiently expressed from the 35S promoter.

Figure 2.

SMXL7 Physically Interacts with D14 in a SL-Dependent Manner. Detection of FRET in N. benthamiana epidermal cells doubly transfected with D14-CFP/SMXL7^d53-YFP, MAX2-CFP/D14-CFP, and MAX2-CFP/SMXL7-YFP pairs, expressed from the 35S promoter. Colocalization of the acceptor and donor molecules is shown in the first and second panel in each row. The CFP donor was excited with using a 405-nm laser line, and emission from the YFP acceptor (indicating the occurrence of FRET) was monitored by FLIM (third panel in each row). The fluorescence lifetime of YFP (green) is shorter than CFP (red). (A) and (B) FRET-FLIM analysis of D14-CFP and SMXL7^d53-YFP in mock treated cells (A) or cells treated with 5 μM rac-GR24 (B) (C) and (D) FRET-FLIM analysis of MAX2-CFP and D14-YFP in mock-treated cells (C) or cells treated with 5 μM rac-GR24 (D). (E) and (F) FRET-FLIM analysis of MAX2-CFP and SMXL7-YFP in mock-treated cells (E) or cells treated with 5 μM rac-GR24 (F). (G) Color scheme for FLIM analysis, indicating fluorescence lifetimes between 3 and 1 ns.

Figure 3.

SMXL7 Variants Affect Protein Stability. (A) Structure of SMXL7 protein identifying conserved domains targeted in this study. (B) to (G) Subcellular localization of SMXL-YFP (B), SMXL^ΔNTP1-YFP (C), SMXL^ΔNTP2-YFP (D), SMXL^ΔEAR-YFP (E), SMXL^alaEAR-YFP (F), and SMXL^{ΔP-loop,ΔEAR}-YFP (G) in N. benthamiana epidermal cells, transiently expressed from the 35S promoter. Bar = 50 μm. (H) to (S) Subcellular localization of SMXL-YFP ([H] and [N]), SMXL^ΔNTP1-YFP ([I] and [O]), SMXL^ΔNTP2-YFP ([J] and [P]), SMXL^ΔEAR-YFP ([K] and [Q]), SMXL^alaEAR-YFP ([L] and [R]), and SMXL^{ΔP-loop,ΔEAR}-YFP ([M] and [S]) in the root tips of homozygous transgenic Arabidopsis lines expressing the fusion proteins from the 35S promoter, treated with 5 μM rac-GR24, at 0 min ([H] to [M]) and 20 min ([N] to [S]) after treatment. Bar = 100 μm.

Figure 4.

SMXL7 Responds to SL Treatment in Leaves and Stems. Red signal is chloroplast fluorescence. Yellow signal is SMXL7-YFP/VENUS. (A) to (H) Response of SMXL7 variants in leaf petioles to 20 min treatment with 5 μM rac-GR24: SMXL7-VENUS in the Col-0 ([A] and [B]) and max2-1 ([C] and [D]) backgrounds; SMXL7^d53-VENUS in Col-0 ([E] and [F]) and SMXL7^d53-YFP in Col-0 ([G] and [H]). Expression from the SMXL7 promoter ([A] to [F]) or 35S promoter ([G] and [H]). (I) Very low levels of SMXL7-VENUS in longitudinal hand sections through Col-0 stems. (J) to (P) Response of SMXL7 variants expressed in longitudinal hand sections of stems to treatment with 5 μM rac-GR24 (treatment time indicated in panel); SMXL7-VENUS in smxl6-4 smxl7-3 smxl8-1 max2-1 (s678m2) (J) and max1-1 ([K] and [L]) backgrounds; SMXL7^d53-VENUS in Col-0 ([M] and [N]); SMXL7^d53-YFP in Col-0 (O) and SMXL7^ΔP-loop-YFP in Col-0 (P). Expression from the SMXL7 promoter ([I] to [N]) or 35S promoter ([O] and [P]). White arrows indicate SMXL7 expressing nuclei before and after treatment with rac-GR24.

Figure 5.

Effect of SMXL7 Dose on Development. (A) to (F) Rosette leaf morphology in Col-0 ([A] and [B]), smxl6-4 smxl7-3 smxl8-1 max2-1 (s678m2) ([C] and [D]), and max2-1 ([E] and [F]) untransformed ([A], [C], and [E]) or homozygous for SMXL7pro:SMXL7-VENUS ([B], [D], and [F]). (G) to (L) Adult shoot morphology in Col-0 ([G] and [H]), smxl6-4 smxl7-3 smxl8-1 max2-1 (s678m2) ([I] and [J]), and max2-1 ([K] and [L]) untransformed ([G], [I], and [K]) or homozygous for SMXL7pro:SMXL7-VENUS ([H], [J], and [L]). (M) Leaf blade length, blade width, and petiole length in the 7th leaf of Col-0, smxl6-4 smxl7-3 smxl8-1 max2-1 (s678m2), and max2-1 untransformed (−) or homozygous for SMXL7pro:SMXL7-VENUS (+). n = 11 to 16; error bars indicate se; bars with the same letter are not significantly different from one another (ANOVA + Tukey HSD, P < 0.05). (N) Number of cauline, rosette and total primary branches in Col-0, smxl6-4 smxl7-3 smxl8-1 max2-1 (s678m2), and max2-1 untransformed (−) or transformed with SMXL7pro:SMXL7-VENUS (+), measured at proliferative arrest. n = 20 to 24; error bars indicate se; bars with the same letter are not significantly different from one another (ANOVA + Tukey HSD, P < 0.05).

Figure 6.

Effect of SMXL7 Stabilization on Development. (A) to (H) Rosette leaf morphology in Col-0 untransformed (A) or transformed with SMXL7pro:SMXL7-VENUS (B), SMXL7pro:SMXL7^d53-VENUS (C), SMXL7pro:SMXL7^ΔP-loop-VENUS (D), 35Spro:SMXL7^d53-YFP (F), 35Spro:SMXL7^ΔP-loop-YFP ([G], hemizygous; [H], homozygous), and in d14-1 (E). (I) to (M) Adult shoot morphology (main picture) and reproductive morphology (inset) in Col-0 transformed with SMXL7pro:SMXL7^d53-VENUS (I), SMXL7pro:SMXL7^ΔP-loop-VENUS (J), 35Spro:SMXL7^d53-YFP (K), and 35Spro:SMXL7^ΔP-loop-YFP ([L], hemizygous; [M], homozygous). (N) Leaf blade length, blade width and petiole length in the 7th leaf of untransformed Col-0, Col-0 transformed with SMXL7pro:SMXL7-VENUS, SMXL7pro:SMXL7^d53-VENUS, SMXL7pro:SMXL7^ΔP-loop-VENUS, 35Spro:SMXL7^d53-YFP, 35Spro:SMXL7^ΔP-loop-YFP (hemizygous), and in d14-1. n = 9 to 16; error bars indicate se; bars with the same letter are not significantly different from one another (ANOVA + Tukey HSD, P < 0.05). (O) Number of cauline, rosette, and total primary branches of untransformed Col-0, Col-0 transformed with SMXL7pro:SMXL7-VENUS, SMXL7pro:SMXL7^d53-VENUS, SMXL7pro:SMXL7^ΔP-loop-VENUS, 35Spro:SMXL7^d53-YFP, 35Spro:SMXL7^ΔP-loop-YFP (hemizygous), and in d14-1. n = 10 to 13; error bars indicate se; bars with the same letter are not significantly different from one another (ANOVA + Tukey HSD, P < 0.05).

Figure 7.

SMXL7 Influences PIN1 and Auxin Transport. (A) to (D) Expression of SMXL7pro:GUS ([A] and [B]) and PIN1pro:GUS ([C] and [D]) in transverse ([A] and [D]) and longitudinal ([B] and [C]) sections of Arabidopsis inflorescence stems. Small bold letters indicate tissues within the stem: C, cambium; XP, xylem parenchyma. (E) Coexpression of SMXL7-VENUS (red, arrowheads) and PIN1-GFP (green) in xylem parenchyma cells of the Arabidopsis inflorescence stem in a line doubly homozygous for the fusion proteins expressed from their respective native promoters. The bright-field image is superimposed, showing cell boundaries and spiral thickening on the adjacent xylem. Blue indicates chloroplast autofluorescence. (F) and (G) Expression of PIN1pro:PIN1-GFP in Col-0 and Col-0 homozygous for SMXL7pro:SMXL7^d53-VENUS. (H) Quantification of PIN1-GFP levels at the basal plasma membrane in xylem parenchyma cells of Arabidopsis inflorescence stems in Col-0 and Col-0 homozygous for SMXL7pro:SMXL7^d53-VENUS. n = 8 plants per genotype, five membranes measured in each plant. Error bars indicate se; asterisks indicate significant difference from the wild type at the P < 0.005 level (t test). (I) Transport of radiolabeled IAA through 18-mm stem segments of Col-0 and Col-0 homozygous for SMXL7pro:SMXL7^d53-VENUS over a 6-h timeframe. n = 17 to 19; error bars indicate se. Asterisks indicate significant difference from the wild type at the P < 0.005 level (t test).

Figure 8.

Phenotypic Effects of SMXL7 Are Partially EAR-Independent. (A) to (F) Rosette leaf morphology in Col-0 (A), max2-1 (B), untransformed smxl6-4 smxl7-3 smxl8-1 max2-1 (s678m2) (C), or s678m2 homozygous for SMXL7pro:SMXL7-VENUS (D), SMXL7pro:SMXL7^alaEAR-VENUS (E), and SMXL7pro:SMXL7^ΔEAR-VENUS (F). (G) Leaf blade length, blade width, and petiole length in the 7th leaf of Col-0, max2-1, untransformed smxl6-4 smxl7-3 smxl8-1 max2-1 (s678m2), or s678m2 homozygous for SMXL7pro:SMXL7-VENUS, SMXL7pro:SMXL7^alaEAR-VENUS, and SMXL7pro:SMXL7^ΔEAR-VENUS. n = 10; error bars indicate se; bars with the same letter are not significantly different from one another (ANOVA + Tukey HSD, P < 0.05). (H) Number of cauline, rosette, and total primary branches of Col-0, max2-1, untransformed smxl6-4 smxl7-3 smxl8-1 max2-1 (s678m2), or s678m2 homozygous for SMXL7pro:SMXL7-VENUS, SMXL7pro:SMXL7^alaEAR-VENUS, and SMXL7pro:SMXL7^ΔEAR-VENUS. n = 10; error bars indicate se; bars with the same letter are not significantly different from one another (ANOVA + Tukey HSD, P < 0.05). (I) Adult shoot morphology in (left to right) Col-0, max2-1, untransformed smxl6-4 smxl7-3 smxl8-1 max2-1 (s678m2), or s678m2 homozygous for SMXL7pro:SMXL7-VENUS, SMXL7pro:SMXL7^ΔEAR–VENUS, and SMXL7pro:SMXL7^alaEAR-VENUS. (J) Height of the primary inflorescence stem in Col-0, max2-1, untransformed smxl6-4 smxl7-3 smxl8-1 max2-1 (s678m2), or s678m2 homozygous for SMXL7pro:SMXL7-VENUS, SMXL7pro:SMXL7^alaEAR-VENUS, and SMXL7pro:SMXL7^ΔEAR-VENUS, measured at proliferative arrest. n = 10; error bars indicate se; bars with the same letter are not significantly different from one another (ANOVA + Tukey HSD, P < 0.05). (K) to (P) Stem anatomy in Col-0 (K), max2-1 (L), untransformed smxl6-4 smxl7-3 smxl8-1 max2-1 (s678m2) (M), or s678m2 homozygous for SMXL7pro:SMXL7-VENUS (N), SMXL7pro:SMXL7^alaEAR-VENUS (O), and SMXL7pro:SMXL7^ΔEAR-VENUS (P) plants stained with toluidine blue. Red bar illustrates the depth of the interfascicular cambium layer. Images are representative of multiple independent samples.

Figure 9.

Loss of the EAR Motif Counteracts SMXL7 Stabilization. (A) to (C) Rosette leaf morphology in untransformed Col-0 (A) or Col-0 transformed with SMXL7pro:SMXL7^d53-VENUS (B) or with SMXL7pro:SMXL7^d53,alaEAR-VENUS (C). (D) to (F) Rosette leaf morphology in untransformed smxl6-4 smxl7-3 smxl8-1 (smxl678) (D) or smxl678 homozygous for SMXL7pro:SMXL7^d53-VENUS (E) or SMXL7pro:SMXL7^d53,alaEAR-VENUS (F). (G) Leaf blade length, blade width, and petiole length in the 7th leaf of Col-0 and smxl678, either untransformed or homozygous for SMXL7pro:SMXL7^d53-VENUS or SMXL7pro:SMXL7^d53,alaEAR-VENUS. n = 10; error bars indicate se; bars with the same letter are not significantly different from one another (ANOVA + Tukey HSD, P < 0.05). (H) Number of cauline, rosette and total primary branches of Col-0 and smxl678, either untransformed or homozygous for SMXL7pro:SMXL7^d53-VENUS or SMXL7pro:SMXL7^d53,alaEAR-VENUS. n = 10; error bars indicate se; bars with the same letter are not significantly different from one another (ANOVA + Tukey HSD, P < 0.05). (I) to (K) Adult shoot morphology in untransformed Col-0 (I) or Col-0 homozygous for SMXL7pro:SMXL7^d53-VENUS (J) or with SMXL7pro:SMXL7^d53,alaEAR-VENUS (K). (L) to (N) Adult shoot morphology in untransformed smxl678 (L) or smxl678 homozygous for SMXL7pro:SMXL7^d53-VENUS (M) or with SMXL7pro:SMXL7^d53,alaEAR-VENUS (N). (O) to (Q) Stem anatomy in untransformed Col-0 (O) or Col-0 homozygous for SMXL7pro:SMXL7^d53-VENUS (P) or SMXL7pro:SMXL7^d53,alaEAR-VENUS (Q) plants stained with toluidine blue. Red bar illustrates the depth of the interfascicular cambium layer. Images are representative of multiple independent samples. (R) to (T) Stem anatomy in untransformed smxl678 (R) or smxl678 homozygous for SMXL7pro:SMXL7^d53-VENUS (S) or SMXL7pro:SMXL7^d53,alaEAR-VENUS (T) plants stained with toluidine blue. Red bar illustrates the depth of the interfascicular cambium layer.

Figure 10.

SMXL7 Activity and EAR Dependency. Summary diagram illustrating the effects of modulating SMXL7 activity on different shoot architectural traits. The smxl678 triple mutant represents a very low level of SMXL7-like activity relative to the wild type, while stabilized SMXL7 represents much higher activity than the wild type, further enhanced when SMXL7 is also overexpressed (orange triangle). Different phenotypes show different sensitivity to modulation of SMXL7 activity. Class I traits, for instance branch angle, are sensitive to SMXL7 across the whole range of activity. Class II traits, for instance blade width, are especially sensitive to the lower range of SMX7 activity, but not to the higher range. Class III traits, including shoot branching and leaf blade length and height, are especially sensitive to the higher range of SMXL7 activity, but not to the lower end of the range. Class IV traits do not show a linear relationship with SMXL7 activity. Phenotypic effects that occur at the higher range of SMXL7 activity are dependent on the presence of the EAR motif, but those at the lower end of the range do not require the EAR motif (purple triangle).