Modification of Serine 1040 of SIBRI1 Increases Fruit Yield by Enhancing Tolerance to Heat Stress in Tomato

Abstract

High temperature is a major environmental factor that adversely affects plant growth and production. SlBRI1 is a critical receptor in brassinosteroid signalling, and its phosphorylation sites have differential functions in plant growth and development. However, the roles of the phosphorylation sites of SIBRI1 in stress tolerance are unknown. In this study, we investigated the biological functions of the phosphorylation site serine 1040 (Ser-1040) of SlBRI1 in tomato. Phenotype analysis indicated that transgenic tomato harbouring SlBRI1 dephosphorylated at Ser-1040 showed increased tolerance to heat stress, exhibiting better plant growth and plant yield under high temperature than transgenic lines expressing SlBRI1 or SlBRI1 phosphorylated at Ser-1040. Biochemical and physiological analyses further showed that antioxidant activity, cell membrane integrity, osmo-protectant accumulation, photosynthesis and transcript levels of heat stress defence genes were all elevated in tomato plants harbouring SlBRI1 dephosphorylated at Ser-1040, and the autophosphorylation level of SlBRI1 was inhibited when SlBRI1 dephosphorylated at Ser-1040. Taken together, our results demonstrate that the phosphorylation site Ser-1040 of SlBRI1 affects heat tolerance, leading to improved plant growth and yield under high-temperature conditions. Our results also indicate the promise of phosphorylation site modification as an approach for protecting crop yields from high-temperature stress.

Keywords: SlBRI1; heating tolerance; phosphorylation site; tomato; yield.

Figure 1

SlBRI1 Ser-1040 influences autophosphorylation of SlBRI1. (A) Alignment of the partial kinase domain sequences of BRI1 homologues. Conserved and similar residues were highlighted with black and gray grounds, respectively. The set of Ser-1040 among BRI1 homologues was marked by the red arrow. Each symbol with different colors at the bottom indicated the conservation of residue at each site. SlBRI1 (Solanum lycopersicum, NP_001296180.1), CsBRI1 (Camelina sativa, XP_010431911.1), CrBRI1 (Camelina sativa, XP_010431911.1), AtBRI1 (Arabidopsis thaliana, NP_195650.1), EsBRI1 (Eutrema salsugineum, XP_006411743.1), BraBRI1 (Brassica oleracea var. oleracea, XP_013597742.1), BnBRI1 (Brassica napus, NP_001303105.1), BrBRI1 (Brassica rapa XP_009101880.2), TcBRI1 (Theobroma cacao, XP_017985424.1), CclBRI1 (Citrus clementina, XP_006427932.1), StBRI1 (Solanum tuberosum, XP_006357355.1), TaBRI1 (Triticum aestivum, DQ_655711.1), ZmBRI1 (Zea mays, XP_008656807.1) and OsBRI1 (Oryza sativa, NP_001044077.1). (B) Autophosphorylation level of SlBRI1 in vitro. Autophosphorylation analysis of recombinant FLAG-SlBRI1, FLAG-K916E, FLAG-S1040A, and FLAG-S1040D proteins was detected by anti-pThr antibodies, and anti-FLAG antibodies were used to show the loading levels for western blotting. Intensities of bands were presented as relative values compared with the FLAG-SlBRI1.

Figure 2

SlBRI1 Ser-1040 slightly affects BR signalling in tomato. (A) Phenotypes of plants at the maturation stage. The plants shown from left to right are as follows: cu3^-abs1, SlBRI1-1, SlBRI1-3, S1040A-8, S1040A-10, S1040D-1, and S1040D-2. (B) Western blot analysis of transgenic protein expression using anti-green fluorescent protein (GFP) antibodies. Ponceau S (Solarbio, P8330, Beijing, China) staining shows loading. (C) and (D) Relative transcript levels of SlBRI1 (C) and the BR signalling marker gene SlCPD (D) in tomato. (E) and (F) Dose-response curves of relative hypocotyl lengths of tomato seedlings grown in the dark for 10 days on the surface of media with increasing concentrations of epi-BL (E) and BRZ (F). The data for (E) and (F) are the means ± SDs of 15 independent biological samples.

Figure 3

Dephosphorylation of Ser-1040 improves tomato yield under heat stress. (A) Phenotypes of the second inflorescence (top), fifth inflorescence (middle) and total yield per plant (bottom). (B) Flower numbers of the second and fifth inflorescences. (C) Fruit number and (D) fruit setting rate of the second and fifth clusters. (E) Early yield and total yield per plant. The different letters indicate significant differences at the 0.05 level. The data for (B) and (E) are the means ± SDs of 10 independent biological samples.

Figure 4

Dephosphorylation of Ser-1040 promotes germination and seedling growth under heat stress. (A) Phenotypes of seedlings treated with or without heat stress for 9 days. (B) Germination rate of plants on the fourteenth day after seeding at 33 °C or 28 °C. (C) Germination potential of plants on the fourth day after seeding at 33 °C or 28 °C. (D) Root length and (E) MDA content of plants on the fourteenth day after seeding at 33 °C or 28 °C. (F) Shoot fresh weights and (G) seedling heights of plants at the four-leaf stage treated with or without heat stress (38 °C/28 °C, day/night) for 12 days. The data for (B) and (E) are the means ± SDs of three replicates, and each replicate had 30 plants. The data for (F) and (G) are the means ± SDs of three independent biological samples. The different letters indicate significant differences at the 0.05 level.

Figure 5

Dephosphorylation of Ser-1040 promotes the heat stress tolerance of seedlings. (A–C) Time course of changes in MDA content (A), ion leakage (B), and proline content (C) in tomato seedlings in response to heat stress. Tomato seedlings at the four-leaf stage were placed into a growth chamber set at 38 °C/28 °C (day/night) for 9 days and then transferred to 25 °C for 3 days. The data are the means ± SDs of at least three independent biological samples. The different letters indicate significant differences at the 0.05 level.

Figure 6

Dephosphorylation of Ser-1040 promotes ROS detoxification in seedlings under heat stress. (A) DAB staining for hydrogen peroxide (H₂O₂) in leaves from tomato seedlings during heat stress treatment. (B–D) Time course of changes in CAT (B), SOD (C), and POD (D) activities in tomato seedlings in response to heat stress. Tomato seedlings at the four-leaf stage were placed into a growth chamber set at 38 °C/28 °C (day/night) for 9 days and then transferred to 25 °C for 3 days. The data for (B) to (D) are the means ± SDs of at least three replicates, and each replicate had 10 plants. The different letters indicate significant differences at the 0.05 level.

Figure 7

Dephosphorylation of Ser-1040 promotes photosynthesis in seedlings under heat stress. (A) Time course of changes in total chlorophyll content in tomato seedlings in response to heat stress. (B) CO₂ assimilation rate in tomato at the flowering stages of the second inflorescence (control) and fourth inflorescence (heated). The data are the means ± SDs of at least 3 replicates, and each replicate had 10 plants. The different letters indicate significant differences at the 0.05 level.

Figure 8

Dephosphorylation of Ser-1040 enhances the expression of tomato defence-related genes under heat stress. (A–J) Transcription levels of CAT1 (A), Cu/Zn-SOD (B), POD1 (C), RBOH1 (D), HSFA2 (E), HSFA3 (F), HSP70 (G), HSP90 (H), WRKY1 (I), and WRKY72 (J) in tomato seedlings in response to heat stress. Tomato seedlings at the four-leaf stage were placed into a growth chamber set at 38 °C/28 °C (day/night) for 9 days and then transferred to 25 °C for 3 days. The data are the means ± SDs of three biological replicates and technical replications.