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The SINA1-BSD1 Module Regulates Vegetative Growth Involving Gibberellin Biosynthesis in Tomato
Tuesday, 2024/09/17 | 08:26:22

Yulin YuanYouhong FanLi HuangHan LuBowen TanChloe RamirezChao XiaXiangli NiuSixue ChenMingjun GaoCankui ZhangYongsheng LiuFangming Xiao

Advanced Science; First published: 27 August 2024; https://doi.org/10.1002/advs.202400995

Abstract

In plants, vegetative growth is controlled by synergistic and/or antagonistic effects of many regulatory factors. Here, the authors demonstrate that the ubiquitin ligase seven in absentia1 (SINA1) mammalian BTF2-like transcription factors, Drosophila synapse-associated proteins, and yeast DOS2-like proteins (BSD1) function as a regulatory module to control vegetative growth in tomato via regulation of the production of plant growth hormone gibberellin (GA). SINA1 negatively regulates the protein level of BSD1 through ubiquitin-proteasome-mediated degradation, and the transgenic tomato over-expressing SINA1 (SINA1-OX) resembles the dwarfism phenotype of the BSD1-knockout (BSD1-KO) tomato plant. BSD1 directly activates expression of the BSD1-regulated gene 1 (BRG1) via binding to a novel core BBS (standing for BSD1 binding site) binding motif in the BRG1 promoter. Knockout of BRG1 (BRG1-KO) in tomato also results in a dwarfism phenotype, suggesting BRG1 plays a positive role in vegetative growth as BSD1 does. Significantly, GA contents are attenuated in transgenic SINA1-OXBSD1-KO, and BRG1-KO plants exhibiting dwarfism phenotype and exogenous application of bioactive GA3 restores their vegetative growth. Moreover, BRG1 is required for the expression of multiple GA biosynthesis genes and BSD1 activates three GA biosynthesis genes promoting GA production. Thus, this study suggests that the SINA1-BSD1 module controls vegetative growth via direct and indirect regulation of GA biosynthesis in tomato.

 

See https://onlinelibrary.wiley.com/doi/10.1002/advs.202400995

 

Figure 2; SINA1 interacts with and ubiquitinates BSD1 at lysine residues K93, K293 and K362. A,B) Y2H assay showing BSD1 interacts with SINAs, as indicated by blue coloration of yeast cells (transformed with the bait pEG202 constructs, prey pJG4-5 constructs, or control empty vector (EV)) grown on the selective medium containing X-Gal. C) In vitro binding assay showing that BSD1 directly interacts with SINAs. Epitope-tagged recombinant protein MBP-BSD1-HA was incubated with six MBP-SINAs or unrelated MBP-RHA1B protein and was captured by anti-HA affinity beads, followed by Western blotting using anti-MBP antibody. D) co-IP assay indicating BSD1 interacts with SINA1/2/3 in plant cells. Appropriate combinations of in planta expression constructs were co-expressed in N. benthamiana leaves as indicated together with 50 µm MG132 for two days. Protein extracts were immunoprecipitated by anti-HA affinity matrix and analyzed by Western blotting using appropriate antibodies. BSD1-Flag was specifically co-immunoprecipitated with SINA1/2/3-HA. E) In vitro ubiquitination of BSD1 by SINA1. In vitro ubiquitination assays were conducted in the presence of ATP with the indicated combinations of proteins. The smear banding pattern recognized by anti-HA antibody indicated BSD1 is specifically ubiquitinated by SINA1 (lane 1, top panel). Anti-Flag indicated total ubiquitination catalyzed by six SINAs (bottom panel). F) SINA1 ubiquitinates BSD1 in an E3-dependent manner. MBP-SINA1C63S (mutant without ubiquitin ligase activity) was included for the ubiquitination assay (line 6), only the BSD1 in the presence of recombinant E1, E2, MBP-SINA1 and FLAG-Ub showed polyubiquitination (lane 1, top panel). Anti-Flag indicated total ubiquitination mediated by SINA1 (line 1, bottom panel). G) SINA1 ubiquitinates BSD1 at residues K93, K293 and K362. Tandem mass spectrometric analysis of ubiquitinated peptides from the BSD1 protein was conducted. Three key peptides were identified: A triply charged peptide at m/z 611.3300 corresponding to the sequence GVKEDLSELKETLTR with a ubiquitin remnant (GlyGly) modification at lysine residue 93 (K93) (upper panel); A triply charged peptide at m/z 906.1040 corresponding to the sequence TKPESDWFGIGTFQAKESAYSPR with a ubiquitin remnant modification at K293 (middle panel); A doubly charged peptide at m/z 828.4452 corresponding to the sequence AVIEEDAPSKIIEK with a ubiquitin remnant modification at K362 (lower panel). The fragmentation ions were annotated on the peptide sequence: b ions labeled in blue and y ions in red. All experiments have been repeated at least twice with similar results.

 

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