The Auxin Response Factor OsARF25 Negatively Regulates Grain Size and Weight in Rice (Oryza sativa L.) by Activating the Expression of SG1 and OsOFP04

Abstract

Grain size and grain weight are critical factors influencing crop yield. In rice (Oryza sativa L.), the auxin response factor (OsARF) family proteins, key components of the auxin signaling pathway, function as transcription factors and play essential roles in regulating various plant growth and development processes, including seed development. Here, we identified that Oryza sativa AUXIN RESPONSE FACTOR 25 (OsARF25) plays an essential role in regulating grain size and grain weight by activating the expression of SHORT GRAIN 1 (SG1) and Oryza sativa OVATE FAMILY PROTEIN 04 (OsOFP04). The osarf25 mutants showed larger grains with increased grain length, grain width, and 1000-grain weight. Furthermore, molecular evidence demonstrated that OsARF25 functions as a transcriptional activator. RNA-seq analysis further identified its target genes SG1 and OsOFP04. In addition, OsARF25 directly binds to the promoters of SG1 and OsOFP04 and activates their expression. Further, the osarf25 mutant exhibited enhanced sensitivity to brassinolide treatment, confirming that the targeting of SG1 and OsOFP04 by OsARF25 mediates BR signaling. Taken together, our study revealed that OsARF25 functions as a regulator of grain length, grain width, and grain weight by participating in the BR signaling pathway, and it has potential value for molecular breeding in rice.

Keywords: BR signaling pathway; OsARF25; grain size; grain weight; rice.

Figure 1

Expression analysis of OsARF25. RT-qPCR analysis of relative expression levels of OsARF25 in different tissues from WT. The number after panicles indicates the length (cm); for example, panicle 1 indicates panicles 0~1 cm in length, panicle 2 indicates panicles 1~2 cm in length, and so on. Data are means (±SD) (n = 3). OsActin was used as an internal control for normalization.

Figure 2

OsARF25 functions as a transcriptional activator. (A) Subcellular localization analysis of OsARF25. The scale bar is 5 μm. (B) OsARF25 process transcriptional activation in rice protoplasts. Data are means (±SD.) (n = 3); significant differences were determined using Student’s t-test: *** p < 0.001.

Figure 3

Bioinformatic analysis of the OsARF protein family in rice. (A) Phylogenetic tree of the OsARF protein family in rice. The tree was constructed using the adjacency method in the MEGA6 software. The bootstrap is set to 1000. (B) Conserved motif analysis of the OsARF proteins in rice.

Figure 4

OsARF25 negatively regulates grain size and grain weight. (A) Sequence analysis of the target sites in WT and OsARF25 knockout lines (osarf25-1/-2). In osarf25-1 and osarf25-2 lines, a 1 bp deletion and 1 bp insertion in the second exon results in premature translation termination. (B) Grain phenotypes of WT and osarf25 mutants shown in scale bars of 10 mm. (C–F) Phenotypic differences in grain traits between WT and osarf25 mutants. Grain length (C), grain width (D), grain thickness (E), and 1000-grain weight (F) of osarf25 mutants and WT were analyzed. Data are means (±SD.) (n = 10); significant differences were determined using Student’s t-test: * p < 0.05, **** p < 0.0001.

Figure 5

Screening of target gene for OsARF25. (A) Volcanic map of the differentially expressed genes. (B) Heat map of DEGs in regulating grain size. (C,D) RT-qPCR validation of SG1 (C) and OsOFP04 (D). Data are means (±SD.) (n = 3); significant differences were determined using Student’s t-test: * p < 0.05.

Figure 6

OsARF25 regulates SG1 and OsOFP04 by directly binding to their promoters. (A) Schematic diagram of predicted AuxREs in proSG1 and proOsOFP04. (B) Western blotting of GST and recombinant OsARF25^123-243-GST proteins. Lane 1: 180kDa Plus Prestained Protein Marker; Lane 2: GST protein; Lane 3: OsARF25^123-243-GST. (C,D) An electrophoretic mobility shift assay (EMSA) of OsARF25^123-243-GST binding to the SG1 (C) and OsOFP04 (D) promoters. OsARF25^123-243-GST was incubated with the 6-FAM-labeled probe in the absence or presence of 2-fold, 5-fold, and 10-fold excesses of the corresponding competitors. (E–G) Dual-luciferase reporter assay of OsARF25 and the promoters of SG1 (F) and OsOFP04 (G) in rice protoplasts, containing the indicated constructs shown on the left (E). Data are means (±SD.) (n = 3); significant differences were determined using Student’s t-test: * p < 0.05, *** p < 0.001.

Figure 7

Proposed model explaining how OsARF25 acts upstream of SG1 and OsOFP04 to regulate grain size and grain weight in rice. OsARF25, a key component of auxin signaling, is thought to activate the expression of both SG1 and OsOFP04 by binding directly to their promoters. OsARF25 is suggested to negatively regulate grain size and grain weight by activating the expression of SG1 and OsOFP04.