A novel mitochondrial DnaJ/Hsp40 family protein BIL2 promotes plant growth and resistance against environmental stress in brassinosteroid signaling

Abstract

Plant steroid hormones, brassinosteroids, are essential for growth, development and responses to environmental stresses in plants. Although BR signaling proteins are localized in many organelles, i.e., the plasma membrane, nuclei, endoplasmic reticulum and vacuole, the details regarding the BR signaling pathway from perception at the cellular membrane receptor BRASSINOSTEROID INSENSITIVE 1 (BRI1) to nuclear events include several steps. Brz (Brz220) is a specific inhibitor of BR biosynthesis. In this study, we used Brz-mediated chemical genetics to identify Brz-insensitive-long hypocotyls 2-1D (bil2-1D). The BIL2 gene encodes a mitochondrial-localized DnaJ/Heat shock protein 40 (DnaJ/Hsp40) family, which is involved in protein folding. BIL2-overexpression plants (BIL2-OX) showed cell elongation under Brz treatment, increasing the growth of plant inflorescence and roots, the regulation of BR-responsive gene expression and suppression against the dwarfed BRI1-deficient mutant. BIL2-OX also showed resistance against the mitochondrial ATPase inhibitor oligomycin and higher levels of exogenous ATP compared with wild-type plants. BIL2 participates in resistance against salinity stress and strong light stress. Our results indicate that BIL2 induces cell elongation during BR signaling through the promotion of ATP synthesis in mitochondria.

Fig. 1

Phenotype and characterization of bil2-1D. bil2-1D mutant showed Brz resistance (a, b). a Hypocotyl elongation of wild-type (WT) and bil2-1D seedlings grown on medium containing 0, 1, and 3 μM Brz in the dark for 7 days. b Hypocotyl length of wild-type (WT) and bil2-1D seedlings grown on medium containing 0, 1 and 3 μM Brz in the dark for 7 days. The results are presented as the mean ± SE (n > 30 seedlings). Triple asterisk indicates significant differences relative to the control at P < 0.001 based on the Student’s t test. c, d Phenotype of wild type and bil2-1D seedlings grown in soil under long-day conditions (16 h light, 8 h dark) for 30 days. Side view (c) and top view (d)

Fig. 2

Novel gene is candidate of bil2-1D mutant. a T-DNA insertion site in bil2-1D is indicated with blue, deletion site with green and the BIL2 gene with a red arrow. b Quantitative real-time PCR analysis of At2g42080 mRNA expression in dark-grown, 3-day-old wild-type (WT) and bil2-1D seedlings. The value was normalized against the expression of the ACTIN2 gene. The error bars indicate standard deviation (n = 3). c Alignment of BIL2 (NP_181738) and its homologs in Arabidopsis thaliana, At3g58020 (NP_191361) and At2g18465 (NP_849977). d Alignment of BIL2 (NP_181738) and its homologs in other plants: Arabidopsis lyrata 1 (XP_002881820), Arabidopsis lyrata 2 (XP_002876451), castor bean Ricinus communis (XP_002530712), soybean Glycine max (XP_003519035), grape Vitis vinifera (XP_002279725), and rice Oryza sativa (NP_001059604). The red box indicates the J domain, and yellow box indicates the HPD motif, which is important for the J domain

Fig. 3

BIL2-OX increased the growth of the hypocotyl, inflorescence and roots. a Hypocotyl elongation of wild-type (WT), bil2-1D, BIL2-OX1 and BIL2-OX2 seedlings grown on medium containing 3 μM Brz in the dark for 4 days. b Hypocotyl length of wild type (WT), bil2-1D, BIL2-OX1 and BIL2-OX2 seedlings grown on medium containing 0, 1 and 3 μM Brz in the dark for 4 days. The results are presented as the mean ± SE (n > 30 seedlings). c Real-time PCR analysis of the BIL2 gene expression in the wild-type (WT), BIL2-OX1 and BIL2-OX2 seedlings grown in the light for 24 days. The value was normalized against the expression of the ACTIN2 gene. The error bars indicate standard deviation (n = 3). d Phenotype of wild-type (WT), BIL2-OX1 and BIL2-OX2 seedlings grown in soil under long-day conditions (16 h light, 8 h dark) for 40 days. e–g Measurements indicating primary inflorescence length (e), secondary inflorescence number (f) and branch number (g) of wild-type (WT), BIL2-OX1 and BIL2-OX2 seedlings grown on soil for 40 days. The results are presented as the mean ± SE (n > 15 plants). h Root elongation of wild-type (WT), BIL2-OX1 and BIL2-OX2 seedlings grown on 1/2MS medium light 14 days. Wild-type (WT) is shown in white, BIL2-OX1 in pink and BIL2-OX2 in orange. i Primary root length of wild-type (WT), BIL2-OX1 and BIL2-OX2 grown on 1/2 MS medium in light for 14 and 21 days. j Lateral root number for seedlings grown in light for 14 days. Triple asterisk indicates significant differences relative to the control at P < 0.001 based on the Student’s t test

Fig. 4

The BIL2-RNAi mutant showed short hypocotyls and short inflorescence. a Real-time PCR analysis of the BIL2 gene expression in the wild-type (WT), BIL2-RNAi1 and BIL2-RNAi2 seedlings grown in the light for 7 days. The value was normalized against the expression of the ACTIN2 gene. The error bars indicate standard deviation (n = 3). b Hypocotyl elongation of wild type (WT) and BIL2-RNAi1 and BIL2-RNAi2 seedlings grown on medium containing 3 μM Brz in the dark for 7 days. c Hypocotyl length of wild-type (WT), BIL2-RNAi1 and BIL2-RNAi2 grown on medium containing 0, 0.3, 1 and 3 μM Brz in the dark for 7 days. The results are presented as the mean ± SE (n > 30 seedlings). Triple asterisk indicates significant differences relative to the control at P < 0.001 based on the Student’s t test. d Phenotype of wild type (WT), BIL2-RNAi1 and BIL2-RNAi2 seedlings grown in soil under long-day conditions (16 h light, 8 h dark) for 40 days

Fig. 5

The BR-responsive gene was regulated in BIL2-OX and BIL2-RNAi. a, b Real-time PCR analysis of the regulated BR gene expression in the wild-type (WT), BIL2-OX1 and BIL2-OX2 seedlings grown in the light for 24 days. c, d Real-time PCR analysis of the regulated BR gene expression in the wild-type (WT), BIL2-RNAi1 and BIL2-RNAi2 seedlings grown in the light for 7 days. All values were normalized against the expression of the ACTIN2 gene. The error bars indicate standard deviation (n = 3)

Fig. 6

BIL2 plays a role downstream of BRI1 in BR signaling. a, b The BIL2-OX suppresses the dwarf phenotype of BR receptor mutant bri1-5, in the dark (a) and in the light (b). The plants of wild-type (WT), bri1-5 and bri1-5 × BIL2-OX seedlings grown on 3 μM Brz medium in the dark for 7 days (a) or in soil for 40 days (b)

Fig. 7

The BIL2 gene is expressed in many plant organs. BIL2::promoter::GUS expression patterns in transgenic Arabidopsis plants. Dark 2- and 3-day-old seedlings on the 1/2 MS medium (a, d) treated with BL 100 nM (b, e) and 3 μM Brz (c, f). Real-time PCR analysis of BIL2 gene expression in wild-type seedlings grown in the light for 10 days and treated with 100 nM of BL and 3 μM of Brz (g). The values were normalized against expression of the ACTIN2 gene. The error bars indicate the standard deviation (n = 3). Light-grown 11-day-old seedlings of the shoot apical meristem (SAM) (h) and root (i). Light-grown 28-day-old seedlings of flower bud (j) and pollen (l). Scale bars 0.5 mm (a–f, h) and 2 mm (i–k)

Fig. 8

BIL2 protein localized in the mitochondria. Subcellular localization of BIL2 in transgenic plants. a BIL2-GFP localization in the root tip. b Mitotracker red dye fluorescence. c Merged image of a and b. Scale bar 10 μm

Fig. 9

ATP production by BIL2 promotes hypocotyl elongation. a Hypocotyl elongation by ATP. Hypocotyl lengths of dark 7-day-old seedlings grown in the absence or presence of 1 μM Brz or ATP (125 and 250 μM). Combinatorial treatments are indicated. b Hypocotyl lengths of dark 7-day-old seedlings grown in the absence or presence of oligomycin (OM) (25 and 50 μM). Twenty-five seedlings per treatment were analyzed in each experiment. The data shown are the mean ± SE. c Total ATP concentration in dark-grown 5-day-old seedlings of wild-type (WT), BIL2-OX1 and BIL2-OX2. The data shown are the mean ± SE of three independent experiments. d, e ATP can increase BR signaling. Real-time PCR analysis of regulated BR gene expression in wild-type (WT) seedlings grown in the dark for 7 days and treated with ATP for 3 h

Fig. 10

BIL2-OX showed resistance against salt stress. a Fresh weight above-ground parts of each plants were divided in four groups (0–5, 5–10, 10–15, and 15–20 mg) and the plant numbers were counted in each group. Individual numbers of the wild-type (WT), BIL2-OX1, BIL2-OX2 and BRI-OX seedlings germinated on half-strength MS medium with (right graphics) or without (left graphics) 125 mM NaCl and under light for 25 days. b BIL2-OX and wild-type (WT) plant phenotypes under salt stress. Photographs of the seedlings were obtained at 25 days after germination. The data shown are the mean ± SE of three independent experiments. Asterisk indicates significant differences relative to the control at P < 0.05 based on Student’s t test

Fig. 11

BIL2-OX showed resistance against strong light stress. a The number of plants was counted using the same method as with the salt resistance analysis explained in Fig. 12. The individual numbers of the wild-type (WT), BIL2-OX1 and BIL2-OX2 seedlings germinated on half-strength MS medium under normal light (left graphics) or strong light (left graphics) for 25 days. b BIL2-OX and wild-type (WT) plant phenotype in strong light stress. Photographs of the seedlings were obtained at 25 days after germination. The data shown are the mean values

Fig. 12

Schematic representation of BIL2 in the mitochondria. BIL2 induces cell elongation through BR signaling to promote ATP synthesis in the mitochondria