Figure 1.
Extremely dwarfed phenotypes of the topp4-1 mutant and map-based cloning strategy of the topp4-1 locus.
(A) Representative 2-week-old wild-type, heterozygous, and homozygous mutant seedlings. (B) Representative 6-week-old plants. (C) A 6-week-old topp4-1 homozygous plant. (D) Flowers from topp4-1 mutant are small with irregular and narrow sepals. (E) Siliques from topp4-1 mutant are twisted with few seeds. (F) Numbers of pollen grains per mature anther are reduced in topp4-1 homozygous plants. (G) Numbers of seeds per mature silique are reduced in topp4-1 homozygous plants. (H) topp4-1 was mapped on chromosome 2. Numbers of recombinants are shown below the markers. (I) Genomic structure of TOPP4. Thick black boxes represent exons and lines between the boxes represent introns. Gray boxes represent untranslated regions. Arrowheads represent three TOPP4 alleles identified through this research (topp4-1) or obtained from other resources (N466328 and SALK_090980). Scale bars = 1 cm. Asterisks in (F) and (G) represent statistic differences based on Student's t test with P<0.05. Error bars in (F) and (G) represent standard error (SE) (n = 30).
Figure 2.
Expression of 35S-topp4-1, pTOPP4-topp4-1, or 35S-TOPP4 in wild-type plants.
(A) Expression of 35S-topp4-1 or pTOPP4-topp4-1 in wild type recapitulates the dwarfed phenotypes of topp4-1. Five-week-old 35S-topp4-1 transgenic plants are shown on the left, and upper right corner is the higher magnitude picture of the #2 transgenic plant. Five-week-old pTOPP4-topp4-1 transgenic plant is shown on the right. (B) qRT-PCR was used to detect relative expression levels of the topp4-1 in three 35S-topp4-1 transgenic lines shown in (A). The expression level of TOPP4 in wild type was set to 1.0. (C) Representative 4-week-old amiR-TOPP4 topp4-1 transgenic lines. The first number 1 or 2 of the lines represents transgenic plants generated by amiR-TOPP4-1 or amiR-TOPP4-2, respectively. (D) qRT-PCR was used to detect the relative expression levels of topp4-1 in three amiR-TOPP4 topp4-1 transgenic lines shown in (C). The expression level of topp4-1 in the topp4-1 mutant was set to 1.0. (E) qRT-PCR was used to detect the relative expression levels of TOPP4 in seven 2-week-old transgenic seedlings overexpressing TOPP4. The expression level in wild type was set to 1.0. (F) Five-week-old transgenic plant overexpressing TOPP4 shows increased inflorescence and enlarged organ size compared to the wild-type plant. One representative line (#7) is shown. Three-week-old wild-type plant (left) and 35S-TOPP4 #7 (right) are shown in the upper left corner. Scale bars = 1 cm in (A) and (F), 0.5 cm in (C). Asterisks in (B), (D) and (E) represent statistic differences based on Student's t test with P<0.05. In (B), (D) and (E), error bars represent SE (n = 3).
Figure 3.
Overexpression of TOPP4 in topp4-1 rescued the severely dwarfed phenotype, and amiRNA lines of TOPP4 showed dwarfed phenotypes.
(A) Representative 6-week-old 35S-TOPP4 topp4-1 transgenic lines. (B) qRT-PCR was used to detect the relative expression levels of TOPP4 in five 35S-TOPP4 topp4-1 transgenic lines shown in (A). The expression level of TOPP4 in wild type was set to 1.0. (C) Representative 5-week-old amiR-TOPP4 transgenic lines. The first number 1 or 2 of the lines represents transgenic plants generated by amiR-TOPP4-1 or amiR-TOPP4-2, respectively. The white arrows indicate partial-sterile siliques. (D) The height of 5-week-old wild type and three amiR-TOPP4 transgenic lines. (E) qRT-PCR was used to detect the relative expression levels of TOPP4 in three amiRNA lines shown in (C). The expression level of TOPP4 in wild type was set to 1.0. (F) The protein levels of TOPP4 in wild type, topp4-1, and three amiRNA lines of TOPP4. The coomassie brilliant blue-stained RbcS protein was used as loading controls. Scale bars = 1 cm in (A) and (C). Asterisks in (B), (D) and (E) represent statistic differences based on Student's t test with P<0.05. In (B), (D) and (E), error bars represent SE (n = 3).
Figure 4.
Tissue-specific expression patterns of the TOPP4 gene.
(A)–(K) GUS activity was detected in various organs of pTOPP4-GUS transgenic plants at different development stages. (A) Root from a 7-day-old transgenic plant. (B) Cotyledons of a 7-day-old seedling. (C) Shoot apical meristem from a 12-day-old seedling. (D) One of the first pair of true leaves from a 3-week-old seedling. (E) One of the second pair of true leaves from a 3-week-old seedling. (F) Stomata from a mature rosette leaf. (G) Trichome from a mature rosette leaf. (H) GUS and safranin-stained 7-µm cross section of a mature rosette leaf. (I) Young flowers from a 4-week-old plant. (J) Mature flower from a 4-week-old plant. (K) Silique from a 4-week-old plant. (L) qRT-PCR was used to detect the relative expression levels of TOPP4 gene in different organs. Rosette leaves (RL), roots (R), young seedlings (YS), stems (S), flower buds (FB), young siliques (Ysi). The expression level of TOPP4 in rosette leaves was set to 1.0. Asterisks represent statistic differences based on Student's t test with P<0.05. Error bars represent SE (n = 3). Scale bars = 100 µm in (A); 1 mm in (B)–(E), and (K); 20 µm in (F) and (H); 200 µm in (G), (I), and (J).
Figure 5.
Subcellular localization of the TOPP4 protein.
(A) Subcellular localization of TOPP4-YFP in Arabidopsis protoplasts indicated that TOPP4 is mainly localized in the nucleus and at the plasma membrane. (B) TOPP4-GFP transiently expressed in pavement cells of a tobacco leaf. BF, bright field; GFP, GFP fluorescence; overlay, merged image. (C) Plasmolysis studies showed that TOPP4-GFP is located on plasma membrane in root cells from 10-day-old 35S-TOPP4-GFP transgenic plants. (D) Immunoblotting detection of the TOPP4 protein in purified plasma membrane fraction of wild-type seedlings. PIN1 protein was used as a positive control. Anti-GFP and anti-PIN1 antibodies were used for detecting TOPP4 and PIN1, respectively. Scale bars = 20 µm in (A); 40 µm in (B) and (C).
Figure 6.
topp4-1 is insensitive to exogenously applied GA3, and GA enhances the TOPP4 protein level through a GA-GID1 pathway.
(A) Four-week-old wild-type, ga1-3, and topp4-1 seedlings sprayed with or without 100 µM GA3. Scale bar = 1 cm. (B) Analysis the expression of GA responsive genes EXP8 and PRE1 in wild type, topp4-1, amiR-TOPP4 #1-1, and N466328 by qRT-PCR. The expression levels in wild type were set to 1.0. Asterisks represent statistic differences based on Student's t test with P<0.05. Error bars represent SE (n = 3). (C) The protein levels of TOPP4 in 2-week-old N466328, and 2-week-old wild-type and topp4-1 seedlings treated with different concentrations of GA3. (D) Analysis of the TOPP4 expression in 2-week-old wild type treated with GA3 by qRT-PCR. The expression level in wild type was set to 1.0. Error bar represents SE (n = 3). (E) The protein levels of TOPP4 in wild-type and gid1a/b/c seedlings treated with different concentrations of GA3. Numbers under lanes in (C) and (E) indicate relative band intensities that were quantified and normalized for each panel. The coomassie brilliant blue-stained RbcS protein was used as loading controls.
Figure 7.
Loss-of-function mutants rga-t2 and gai-t6 can partially suppress the dwarfed phenotype of topp4-1.
(A) Representative 5-week-old wild-type, topp4-1, rga-t2, rga-t2 topp4-1, gai-t6, gai-t6 topp4-1, rga-t2 gai-t6, and rga-t2 gai-t6 topp4-1 plants. The rga-t2 topp4-1, gai-t6 topp4-1, and rga-t2 gai-t6 topp4-1 double and triple mutants were back-crossed with Col six times. The rga-t2, gai-t6 single mutants and the rga-t2 gai-t6 double mutant screened from the same genetic cross were used as control. Scale bars = 1 cm. (B) The height of 6-week-old wild-type, rga-t2, gai-t6, rga-t2 gai-t6, topp4-1, rga-t2 topp4-1, gai-t6 topp4-1, and rga-t2 gai-t6 topp4-1 plants. Asterisk represents statistic differences based on Student's t test with P<0.05. Error bars represent SE (n = 20).
Figure 8.
TOPP4 controls the stability of DELLA proteins.
(A) The protein levels of GFP-RGA and GAI in wild-type, topp4-1, and TOPP4 overexpressing plants determined by immunoblotting using antibody against GFP or GAI. (B) The protein levels of RGA in wild-type, topp4-1, and three amiR-TOPP4 plants. (C) In vitro degradation of GFP-RGA. Total protein extractions from wild-type and topp4-1 plants were incubated with TOPP4, topp4-1, or 100 µM MG132 for the indicated time. (D) The degradation of GFP-RGA in wild-type and topp4-1 plants induced by 100 µM GA3 or 100 µM GA3 together with 50 µM CHX. The time refers to the period that seedlings were treated with GA3 or GA3 and CHX before immunoblotting analysis. Numbers under lanes indicate relative band intensities that were quantified and normalized for each panel. The coomassie brilliant blue-stained RbcS protein was used as loading controls.
Figure 9.
TOPP4 can interact with RGA and GAI both in vitro and in vivo.
(A) Pull-down assays were performed to determine the interactions between GST-TOPP4 and HIS-RGA or HIS-GAI. Co-precipitated HIS-RGA or HIS-GAI was analyzed by anti-HIS antibody. (B) Pull-down assay was used to test the competitive binding to GAI between TOPP4 and topp4-1 proteins. GST bound GAI protein was analyzed by anti-HIS antibody. Numbers under lanes indicate relative band intensities quantified and normalized for each panel. (C) Yeast two-hybrid assays were used to determine the interactions between TOPP4/topp4-1 and RGA or GAI. Quantitative measurements of β-gal activities are shown on the right. Error bars represent SE (n = 3). Mutated topp4-1 seemed to interact with RGA or GAI slightly more than did the wild-type TOPP4. (D) Co-immunoprecipitation of RGA or GAI with TOPP4-GFP. Total protein extracts of 35S-TOPP4-GFP plants were immunoprecipitated with an anti-GFP antibody and detected by immunoblotting using antibody against RGA or GAI. Immunoprecipitation by anti-GFP in Col was used as a negative control. (E) BiFC analyses of TOPP4-RGA and TOPP4-GAI interactions in Nicotiana benthamiana epidermal cells. BF, bright field; YFP, YFP fluorescence; DAPI, DAPI staining; Merge, merged view of the YFP and DAPI images. Red arrows in BF indicate the location of nucleus. Negative control without YFP fluorescence is shown on the left.
Figure 10.
DELLA proteins are direct substrates of TOPP4.
(A) Immunoblotting assays of GFP-RGA and GAI proteins incubated with denatured CIP (de-CIP) or CIP. (B) Immunoblotting assays of GFP-RGA and GAI proteins incubated with GST-topp4-1 or GST-TOPP4 from E. coli suggested that TOPP4, but not topp4-1, can directly dephosphorylate phosphorylated RGA and GAI. Phosphorylated status, +P; dephosphorylated status, −P. (C) 2-DE analyses of post-translational modification of GFP-RGA in wild-type, topp4-1, and TOPP4 overexpressing plants. The phosphorylation status of RGA was increased in topp4-1 while the dephosphorylation status of RGA was increased in 35S-TOPP4 plants compared to wild type. The total protein extractions were separated by 2-DE and immunoblotted with anti-GFP antibody. (D) A current model of TOPP4 function on DELLA stability in the GA signaling pathway.