OsRELA Regulates Leaf Inclination by Repressing the Transcriptional Activity of OsLIC in Rice

Abstract

Leaf angle is one of the most important agronomic traits in rice, and changes in leaf angle can alter plant architecture to affect photosynthetic efficiency and thus determine grain yield. Therefore, it is important to identify key genes controlling leaf angle and elucidate the molecular mechanisms to improve rice yield. We obtained a mutant rela (regulator of leaf angle) with reduced leaf angle in rice by EMS mutagenesis, and map-based cloning revealed that OsRELA encodes a protein of unknown function. Coincidentally, DENSE AND ERECT PANICLE 2 (DEP2) was reported in a previous study with the same gene locus. RNA-seq analysis revealed that OsRELA is involved in regulating the expression of ILI and Expansin family genes. Biochemical and genetic analyses revealed that OsRELA is able to interact with OsLIC, a negative regulator of BR signaling, through its conserved C-terminal domain, which is essential for OsRELA function in rice. The binding of OsRELA can activate the expression of downstream genes repressed by OsLIC, such as OsILI1, a positive regulator of leaf inclination in rice. Therefore, our results suggest that OsRELA can act as a transcriptional regulator and is involved in the regulation of leaf inclination by regulating the transcriptional activity of OsLIC.

Keywords: OsLIC; OsRELA; leaf inclination; phytohormone; rice.

FIGURE 1

Phenotypic characterization of the rela mutant. (A) The morphological phenotypes of the wild-type and rela at the grain-filling stage. Bar = 20 cm. WT, wild-type. (B) Quantification of the leaf inclination of the second lamina joint of wild-type and rela. The picture at top shows the second lamina joints. Error bars are SD (n = 30). Asterisks indicate significant differences from the WT (**P < 0.01, Student’s t-test). (C) Transverse section of the lamina joints of wild-type and rela. The red box indicates the enlarged abaxial sides of the lamina joint. Bars = 250 μm in the upper panel and 30 μm in the lower panel. (D) Longitudinal section of the lamina joints of wild-type and rela. The red box indicates the enlarged adaxial sides of the lamina joint. Bars = 250 μm in the upper panel and 30 μm in the lower panel. (E) Measurement of lamina joint adaxial cell lengths of wild-type and rela [shown in (D)]. Error bars are SD (n = 60). Asterisks indicate significant differences from the WT (**P < 0.01, Student’s t-test). (F) The number of sclerenchyma cell layers in the adaxial sides of wild-type and rela [shown in (C)]. Error bars are SD (n = 30). Asterisks indicate significant differences from the WT (**P < 0.01, Student’s t-test). (G) Measurement of sclerenchyma cell radius in the abaxial sides of wild-type and rela [shown in (C)]. Error bars are SD (n = 30). Asterisks indicate significant differences from the WT (**P < 0.01, Student’s t-test).

FIGURE 2

Map-based cloning of OsRELA and complementation test. (A) Linkage map of the OsRELA locus. OsRELA is located on the long arm of chromosome 7, between the molecular marker SNP 25.338 M and Indel 25.402 M, a genomic region of ∼64 kb containing six ORFs. The markers, numbers of recombinants and candidate genes are indicated. (B) Schematic diagram of the OsRELA locus. Comparison with the wild-type sequence revealed a 28-bp deletion (dashed) in the seventh exon, generating a premature stop codon (bold italics). (C) Protein levels of OsRELA in the seedlings of wild-type and rela detected by immunoblot using anti-OsRELA–specific polyclonal antibodies. Ponceau staining of the Rubisco large subunit is shown as a loading control. Molecular masses of proteins (kDa) are shown on the left. (D) Plant morphology of wild-type, rela/proOsRELA:OsRELA(CO), rela and RELA-CRISPR at the heading stage. Bar = 10 cm. (E) Quantification of the leaf inclination of the second lamina joint of wild-type, rela/proOsRELA:OsRELA (CO), rela and RELA-CRISPR plants. Error bars are SD (n = 20). Asterisks indicate significant differences from the WT (**P < 0.01, Student’s t-test).

FIGURE 3

Subcellular localization and tissue expression patterns of OsRELA. (A) Subcellular localization of the OsRELA-GFP fusion protein in rice protoplasts. mCherry-NLS was used as the nuclear marker. Bar = 20 μm. (B) Fluorescent signals in transgenic rice plants expressing GFP-OsRELA. The nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI). Bar = 80 μm. (C) RT–qPCR analysis of the expression patterns of OsRELA in various tissues, including root, stem, leaf blade, lamina joint, leaf sheath and young panicle. The rice UBQ gene was amplified as the internal control. (D) Examination of GUS activity in transgenic plants expressing proOsRELA:GUS. GUS activity is found in the lamina joint, young florets, stem, vascular bundles, leaf sheath and root.

FIGURE 4

RNA-seq analysis of OsRELA-modulated genes. (A) A volcano plot illustrating differentially expressed genes from RNA-seq analysis between wild-type and rela. Genes upregulated and downregulated are shown in red and green, respectively. Values are presented as the log2 of tag counts. (B) Heat map of the RNA-seq analysis results shows all genes that were significantly differentially expressed (left panel) and the representative top 50 genes that were differentially expressed (right panel). (C) Gene ontology (GO) functional clustering of all genes that were differentially expressed. (D) RT–qPCR validation analysis of the gene expression levels between wild-type and rela revealed by RNA-seq. The rice UBQ gene was amplified as the internal control. Asterisks indicate significant differences from the WT (*P < 0.05, **P < 0.01, ***P < 0.001, Student’s t-test). (E) RT–qPCR analysis of the genes involved in cell wall synthesis. The rice UBQ gene was amplified as the internal control. Asterisks indicate significant differences from the WT (**P < 0.01, ***P < 0.001, Student’s t-test).

FIGURE 5

OsRELA physically interacts with and acts downstream of OsLIC. (A) Interactions between OsRELA and OsLIC in yeast two-hybrid assays. OsRELA could not form homodimer in yeast. AD, activation domain; BD, binding domain; SD, synthetic dropout; the gradients indicate tenfold serial dilutions. (B) BiFC analysis of OsRELA and OsLIC interactions in rice protoplasts. BF, brightfield. Scale bars, 5 μm. (C) LIC-GST proteins can pull down OsRELA from total protein extracts of 1-week-old wild-type plants. OsRELA was detected by immunoblotting using anti-OsRELA-specific polyclonal antibodies. (D) Plant morphologies of wild-type, rela, rela/lic-2 and lic-2 at the heading stage. Bar = 10 cm. (E) RT–qPCR analysis of OsILI1 expression levels in wild-type, rela, rela/lic-2 and lic-2 lamina joints. The rice UBQ gene was amplified as the internal control. Asterisks indicate significant differences from the WT (**P < 0.01, Student’s t-test).

FIGURE 6

The OsRELA-OsLIC interaction occurs via the conserved C-terminal of OsRELA. (A) Schematic representation of wild-type and different truncated forms of OsRELA. (B) Yeast two-hybrid assays show that OsLIC interacts with OsRELA and OsRELA-T3. AD, activation domain; BD, binding domain; SD, synthetic dropout; the gradients indicate tenfold serial dilutions. (C) Schematic diagrams of the reporter and effector constructs. The firefly luciferase (LUC) gene driven by the OsILI1 promoter and the Renilla luciferase (REN) reporter gene driven by the 35S promoter were used as the reporter and internal control, respectively. For the effectors, OsRELA, OsRELAΔC and OsLIC were fused with FLAG. (D) Transient gene expression assays in rice protoplasts. The LUC reporter gene was cotransfected with OsRELA, OsRELAΔC and OsLIC or both. Asterisks indicate significant differences from the control (*P < 0.05, **P < 0.01, Student’s t-test). (E) Plant morphologies of wild-type, rela, rela/proOsRELA:OsRELA(CO) and rela/proOsRELA:OsRELAΔC(CO–ΔC) at the heading stage. Bar = 10 cm. (F) RT–qPCR analysis of OsILI1 expression levels in wild-type, rela, rela/proOsRELA:OsRELA(CO) and rela/proOsRELA:OsRELAΔC(CO–ΔC) lamina joints. The rice UBQ gene was amplified as the internal control. Asterisks indicate significant differences from the WT (**P < 0.01, Student’s t-test).

FIGURE 7

A model for OsRELA in regulating the lamina inclination. OsRELA positively regulates the expression of OsIIP3, OsILI5/OsBUL1, OsILI4/OsBU1, and OsILI1, which are positive regulators involved in the lamina inclination. Since it has been reported that OsBZR1 and OsLIC are pair of antagonistic transcription factors that involved in the lamina inclination by regulate the expression of OsILI1. The binding of OsRELA can activate the expression of OsILI1 repressed by OsLIC as well. Whether the OsRELA directly or indirectly activates the listed gene expressions (dashed arrows) should be elucidated in the future.