Modification of Threonine-1050 of SlBRI1 regulates BR Signalling and increases fruit yield of tomato

Abstract

Background: Appropriate brassinosteroid (BR) signal strength caused by exogenous application or endogenous regulation of BR-related genes can increase crop yield. However, precise control of BR signals is difficult and can cause unstable effects and failure to reach full potential. Phosphorylated BRASSINOSTEROID INSENSITIVE1 (BRI1), the rate-limiting receptor in BR signalling, transduces BR signals, and we recently demonstrated that modifying BRI1 phosphorylation sites alters BR signal strength and botanical characteristics in Arabidopsis. However, the functions of such phosphorylation sites in agronomic characteristics of crops remain unclear.

Results: In this work, we investigated the roles of tomato SlBRI1 threonine-1050 (Thr-1050). SlBRI1 mutant cu3^-abs1 plants expressing SlBRI1 with a non-phosphorylatable Thr-1050 (T1050A), with a wild-type SlBRI1 transformant used as a control, were examined. The results showed enhanced autophosphorylation of SlBRI1 and BR signal strength for cu3^-abs1 harbouring T1050A, which promoted yield through increased plant expansion, leaf area, fruit weight and fruit number per cluster but reduced nutrient contents, including ascorbic acid and soluble sugar levels. Moreover, plant height, stem diameter, and internodal distance were similar between the transgenic plants.

Conclusion: Our results reveal the biological role of Thr-1050 in tomato and provide a molecular basis for establishing high-yield crops by precisely controlling BR signal strength via phosphorylation site modification.

Keywords: Agronomic trait; BR signal; Phosphorylation site; SlBRI1; Tomato.

Fig. 1

Dephosphorylation of Thr-1050 improves plant vegetative growth. a Top, western blot analysis of transgenic protein expression using anti-green fluorescent protein (GFP) antibodies. CBB, Coomassie brilliant blue. Bottom, phenotypes of plants at the germination stage (8 days after sowing), seedling stage (40 days after sowing), and maturation stage (120 days after sowing). b Relative transcript levels of SlBRI1 by qRT-PCR. c Plant height of plants at the maturation stage. d Internodal length and (e) stem diameters of the second flower node. f Plant expansion at the maturation stage. g Stem-leaf angle, (h) ultrastructure by scanning electron microscopic under a 1500x-magnified visual field, and (i) leaf area of the sixth leaf. j Cell number per unit of the sixth leaf under a 300x-magnified visual field. Data are the means ± SDs of at least 5 independent biological samples. Asterisks indicate significant differences compared with P_SlBRI1::SlBRI1-GFP plants (*P < 0.05; **P < 0.01; Student’s t-test)

Fig. 2

Dephosphorylation of Thr-1050 promotes tomato yield. a Top, fruits from the first to the fourth fruit node. Middle, phenotypes of the third inflorescence. Bottom, phenotypes of the third cluster. b Fruit yield per plant. c Individual fruit weight at the red ripening (RR) stage. d Pericarp thickness of the fruit at the yellow ripening stage. e Seed number per fruit. f Thousand-seed weight. Data are the means ± SDs of 15 independent biological samples. Asterisks indicate significant differences compared with P_SlBRI1::SlBRI1-GFP plants (*P < 0.05; **P < 0.01; Student’s t-test)

Fig. 3

Dephosphorylation of Thr-1050 alters fruit quality. a Fruit shape index at the breaker stage. b Fruit firmness at yellow ripening (YR) and red ripening (RR) stages. c and (d) Contents of total ascorbic acid (AsA) (c) and reduced AsA (d). e, (f), (g) and (h) Contents of fructose (e), glucose (f), malic acid (g), and citric acid (h) measured using HPLC. Date for (a) and (b) are the means ± SDs of 15 independent biological samples; data for (c) to (h) are the means ± SDs of 3 independent biological samples. FW = fresh weight. Asterisks indicate significant differences compared with P_SlBRI1::SlBRI1-GFP plants (*P < 0.05; **P < 0.01; Student’s t-test)

Fig. 4

Dephosphorylation of Thr-1050 affects BR signalling. a and (b) Relative transcript levels of BR signalling marker genes SlCPD and SlDWARF were tested by qRT-PCR. c BR content in the third leaf measured using a BR ELISA Kit. Data are the means ± SDs of 3 independent biological samples. d Hypocotyl length of seedlings grown in the dark for 9 days on the surface of the medium. e Dose-response curves of relative hypocotyl length of seedlings grown in the dark for 9 days on the surface of media supplemented with increasing concentrations of epibrassinolide (epi-BL). Data for (d) and (e) are the means ± SDs of 15 independent biological samples. Asterisks indicate significant differences compared with P_SlBRI1::SlBRI1-GFP plants (*P < 0.05; **P < 0.01; Student’s t-test)

Fig. 5

Dephosphorylation of Thr-1050 influences SlBRI1 autophosphorylation. a Autophosphorylation level of SlBRI1 in vitro. Autophosphorylation activity of recombinant FLAG-SlBRI1, FLAG-T1050A, FLAG-T1050D, and FLAG-K916E proteins was detected using anti-pThr antibodies, and aliquots of the recombinant proteins were detected using anti-FLAG antibodies and western blotting. Coomassie brilliant blue (CBB) staining shows loading. b The relative autophosphorylation levels of FLAG-SlBRI1, FLAG-T1050A, FLAG-T1050D, and FLAG-K916E proteins in vitro. The autophosphorylation level of FLAG-SlBRI1 was defined as “1”. Data are the means ± SDs of 3 independent measurements. c Autophosphorylation level of SlBRI1 in vivo. Autophosphorylation activity of SlBRI1-green fluorescent protein (GFP), T1050A-GFP, T1050D-GFP, and K916E-GFP proteins was detected using anti-pThr antibodies; anti-GFP antibodies were used to show the loading levels for western blotting. d The relative autophosphorylation levels of SlBRI1-GFP, T1050A-GFP, T1050D-GFP, and K916E-GFP proteins in vivo. The autophosphorylation level of SlBRI1-GFP was defined as “1”. Data are the means ± SDs of 3 independent measurements