A Mitochondrial Localized Chaperone Regulator OsBAG6 Functions in Saline-Alkaline Stress Tolerance in Rice

Abstract

B-cell lymphoma 2 (Bcl-2)-associated athanogene (BAG) family genes play prominent roles in regulating plant growth, development, and stress response. Although the molecular mechanism underlying BAG's response to abiotic stress has been studied in Arabidopsis, the function of OsBAG underlying saline-alkaline stress tolerance in rice remains unclear. In this study, OsBAG6, a chaperone regulator localized to mitochondria, was identified as a novel negative regulator of saline-alkaline stress tolerance in rice. The expression level of OsBAG6 was induced by high concentration of salt, high pH, heat and abscisic acid treatments. Overexpression of OsBAG6 in rice resulted in significantly reduced plant heights, grain size, grain weight, as well as higher sensitivity to saline-alkaline stress. By contrast, the osbag6 loss-of-function mutants exhibited decreased sensitivity to saline-alkaline stress. The transcriptomic analysis uncovered differentially expressed genes related to the function of "response to oxidative stress", "defense response", and "secondary metabolite biosynthetic process" in the shoots and roots of OsBAG6-overexpressing transgenic lines. Furthermore, cytoplasmic levels of Ca²⁺ increase rapidly in plants exposed to saline-alkaline stress. OsBAG6 bound to calcium sensor OsCaM1-1 under normal conditions, which was identified by comparative interactomics, but not in the presence of elevated Ca²⁺. Released OsCaM1-1 saturated with Ca²⁺ is then able to regulate downstream stress-responsive genes as part of the response to saline-alkaline stress. OsBAG6 also interacted with energy biosynthesis and metabolic pathway proteins that are involved in plant growth and saline-alkaline stress response mechanisms. This study reveals a novel function for mitochondrial localized OsBAG6 proteins in the saline-alkaline stress response alongside OsCaM1-1.

Keywords: Chaperone regulator; Grain size; Plant height; Rice; Saline-alkaline stress.

Fig. 1

Multiple alignment of amino acid sequence and phylogenetic analysis of BAG6 orthologs in plants. A Multiple alignment of amino acid sequence of OsBAG6 and its orthologs in other species. The IQ motif and BAG domain are shaded in different colors. B The phylogenetic tree was constructed by Maximum Likelihood method using MEGA X software using the amino acid sequences of the proteins. The sequences of the proteins were obtained from Phytozome 13 (https://phytozome-next.jgi.doe.gov/). The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. Monocots or dicotyledons are shaded in yellow or blue colors, respectively

Fig. 2

Subcellular localization and spatial and temporal expression patterns of OsBAG6. A Expression patterns of OsBAG6 under abiotic stress and ABA treatment. OsGAPDH1 was used as the internal control. Error bars indicate ± SD (n = 3). B Relative expression level of OsBAG6 in the indicated tissues of Kitaake plants, as determined by real-time quantitative PCR (RT-qPCR). OsGAPDH1 was used as the internal control. Error bars indicate ± SD (n = 3). C Subcellular localization analysis of OsBAG6. OsBAG6-GFP was co-transfected with OsCOX11-mCherry plasmid into rice (Kitaake) protoplasts. GFP, green fluorescent protein; RFP, red fluorescent protein. Scale bar = 10 μm

Fig. 3

Characterization of OsBAG6 overexpression lines under saline-alkaline stress conditions. Image (A) and survival rates (B) of Kitaake and 3 independent OsBAG6OE lines before and after recovery from saline-alkaline stress condition (25 mM Na₂CO₃, pH = 10.0). In A, scale bar = 8 cm. In B, Data represent mean ± SD of 5 replicates (n = 5, 32 plants per genotype were used to calculate survival rate per replicate). Significant differences were evaluated by two-way ANOVA, followed by Tukey’s multiple comparison test. C DAB staining of leaves of indicated genotypes before and after 24 h saline-alkaline stress (25 mM Na₂CO₃, pH = 10.0). Three biological replicates were performed, with 25 plants per treatment

Fig. 4

Characterization of osbag6 mutant lines under saline-alkaline stress conditions. A Sequence chromatograms showing mutations introduced in the OsBAG6 gene in the osbag6-1 and osbag6-2 mutants using CRISPR/Cas9 technology, as revealed by Sanger sequencing. B Isolation of Cas9-free mutants. The indicated genotypes were sown in media including 50 mg L⁻¹ hygromycin. C Immunoblot analysis of OsBAG6-FLAG in Kitaake, osbag6-1, osbag6-2, and 2 independent complementation lines (Com#1 and Com#2) using anti-FLAG antibody. Histone H3 was used as a loading control. Image (D) and survival rate (E) of Kitaake, 2 independent osbag6 mutant lines, Com#1 and Com#2 before and after recovery from saline-alkaline stress condition (25 mM Na₂CO₃, pH = 10.0). In D, scale bar = 8 cm. In E, Data represent mean ± SD of 5 replicates (n = 5, 32 plants per genotype were used to calculate survival rate per replicate). Significant differences were evaluated by two-way ANOVA, followed by Tukey’s multiple comparison test

Fig. 5

Effect of saline-alkaline stress on Na, Fe, Zn, and Mn in the shoots and roots. A–D The concentration of Na, Fe, Zn, and Mn in the shoots of kitaake, osbag6 mutants and OsBAG6 complementation lines under normal and 25 mM Na₂CO₃ treatment conditions. E–H The concentration of Na, Fe, Zn, and Mn in the roots of kitaake, osbag6 mutants and OsBAG6 complementation lines under normal and 25 mM Na₂CO₃ treatment conditions. Data represent mean ± SD of 3 replicates (20 plants per genotype were used for sampling). Significant differences were evaluated by two-way ANOVA

Fig. 6

Agronomic traits of OsBAG6 overexpression (OsBAG6OE) lines. A Photographs of 65-day-old Kitaake, OsBAG6OE-1, OsBAG6OE-2 and OsBAG6OE-3 plants in a green house. Scale bar = 13 cm. B Plant height of the indicated genotypes. Data represent mean ± SD (n = 20 plants per genotype). C Heading date of the indicated genotypes. Data represent mean ± SD (n = 20 plants per genotype). Image (D) and measurements (E) of grain length in the indicated genotypes. Scale bar = 1 cm, Data represent mean ± SD of 10 biological replicates. Image (F) and measurements (G) of grain width in the indicated genotypes. Scale bar = 1 cm, Data represent mean ± SD of 10 biological replicates. H Measurements of 100-grain weight in the indicated genotypes. Data represent mean ± SD of 10 biological replicates (each contains 100 grains). Significant differences were evaluated by one-way ANOVA, followed by Tukey’s multiple comparison test, ** represent p < 0.01

Fig. 7

OsBAG6 affects the landscape of transcriptional regulation in rice. A Hierarchical clustering analysis of differentially expressed genes (DEGs) in shoot: upregulated genes (URGs) and downregulated genes (DRGs) between wild-type (Kitaake) and OsBAG6OE. High expression level displayed as red color, and blue indicates low expression. Color scale is shown base on Log₂(FPKM). B Gene ontology (GO) enrichment analysis of DEGs was performed to categorize function of URGs and DRGs in shoot. C Hierarchical clustering analysis of DEGs in root: URGs and DRGs between Kitaake and OsBAG6OE. High expression level displayed as red color, and blue indicates low expression. Color scale is shown base on Log₂(FPKM). D GO enrichment analysis of DEGs was performed to categorize function of URGs and DRGs in root

Fig. 8

OsBAG6 interacts with OsCaM1-1 both in vivo and in vitro. A Venn diagram showing overlap of proteins identified by affinity purification and mass spectrometry analysis in two repeats. B OsBAG6 binding proteins identified by IP-MS. The percentage of full-length protein covered by identified unique peptides were defined as Coverage. Unique peptides indicate the number of identified peptides that are mapped to an individual protein. C GO enrichment analysis of 276 genes shade in red encoding OsBAG6 and OsBAG6 interacting proteins in Fig. 8A. D Immunoblot analysis of the results from the co-immunoprecipitation (co-IP) assay. OsBAG6-FLAG construct was co-transfected with OsCaM1-1-HA into Kitaake protoplasts. OsBAG6-FLAG and OsCaM1-1-HA were separately transfected as negative control. Anti-FLAG antibody was used to perform immunoprecipitation, and pulled-down proteins were detected using anti-HA antibody. E Yeast two hybrid (Y2H) assay was performed to test the binding between OsBAG6 with OsCaM1-1 in vitro. F Pull-down assay was used to test the binding affinity between OsBAG6 with OsCaM1-1 in the presence of 5 mM EGTA or 2 mM Ca²⁺. Magnetic beads attaching anti-FLAG antibody was used for pull-down