Rice GROWTH-REGULATING FACTOR7 Modulates Plant Architecture through Regulating GA and Indole-3-Acetic Acid Metabolism

Abstract

Plant-specific GROWTH-REGULATING FACTORs (GRFs) participate in central developmental processes, including leaf and root development; inflorescence, flower, and seed formation; senescence; and tolerance to stresses. In rice (Oryza sativa), there are 12 GRFs, but the role of the miR396-OsGRF7 regulatory module remains unknown. Here, we report that OsGRF7 shapes plant architecture via the regulation of auxin and GA metabolism in rice. OsGRF7 is mainly expressed in lamina joints, nodes, internodes, axillary buds, and young inflorescences. Overexpression of OsGRF7 causes a semidwarf and compact plant architecture with an increased culm wall thickness and narrowed leaf angles mediated by shortened cell length, altered cell arrangement, and increased parenchymal cell layers in the culm and adaxial side of the lamina joints. Knockout and knockdown lines of OsGRF7 exhibit contrasting phenotypes with severe degradation of parenchymal cells in the culm and lamina joints at maturity. Further analysis indicated that OsGRF7 binds the ACRGDA motif in the promoters of a cytochrome P450 gene and AUXIN RESPONSE FACTOR12, which are involved in the GA synthesis and auxin signaling pathways, respectively. Correspondingly, OsGRF7 alters the contents of endogenous GAs and auxins and sensitivity to exogenous phytohormones. These findings establish OsGRF7 as a crucial component in the OsmiR396-OsGRF-plant hormone regulatory network that controls rice plant architecture.

Figure 1.

Phenotypic characterization. A, Gross morphology of wild-type (WT), GRF7OE-1, and GRF7RNAi-1 plants. Bars = 15 cm. B, Comparison of the flag leaf angles (top row) and the top second leaf angles (bottom row) between OsGRF7 transgenic lines. Bars = 1 cm. C, Transverse (top row) and longitudinal (bottom row) sections of internodes in wild-type, GRF7OE-1, and GRF7RNAi-1 plants. Bars = 200 μm. D, Cell length of the internode. E, Culm wall thickness of OsGRF7 transgenic lines. F, Transverse (top row) and longitudinal (bottom row) sections of the lamina joint of flag leaves. Red boxed areas in the top row are enlarged in the middle row. Red arrows indicate adaxial side cell layers measured in G. ad, Adaxial surface of the lamina joint; pc, parenchymal cell; vb, vascular bundle. Bars = 200 μm. G, Adaxial side cell layers of the transverse sections of lamina joints. In D, E, and G, values are means ± sd of five biological replicates. Asterisks indicate significant difference by two-tailed Student’s t test (***P < 0.001).

Figure 2.

OsGRF7 is mainly repressed by OsmiR396e and expressed in various tissues. A, The OsGRF7 cleavage site sequence complementary to OsmiR396. The position corresponding to the 5′ end of the cleaved OsGRF7 mRNA determined by 5′ RACE (rapid amplification of cDNA end) and the frequency of 5′ RACE clones corresponding to the cleavage site is shown by the arrow. 8/12 means eight of 12 clones have an OsmiR396 cleavage site. The mutated sites in OsrGRF7 are marked in red. The asterisks indicate mismatched sites between OsmiR396 and OsGRF7. The gray box indicates the QLQ domain, the pink box indicates the WRC domain, and the purple box indicates the C-terminal end of OsGRF7. B, Protein immunoblotting of OsGRF7 coexpressed with OsmiR396s in GRF7-GFP transgenic rice protoplasts. Vector and control mean with or without the empty vector, respectively. Coomassie Brilliant Blue (CBB) staining was used as a loading control. The relative protein amounts were determined by ImageJ (National Institutes of Health). C, Relative expression levels of OsGRF7 and OsmiR396s were detected by RT-qPCR in internode, root, inflorescence (10 cm), node, flag leaf, shoot apical meristem (SAM), axillary bud, and lamina joint. D, GRF7pro:GUS expression patterns in transgenic plants. Images are as follows: 1, small panicle branch; 2 to 4, inflorescence at different stages; 5 and 6, spikelet; 7, root tip; 8, flag leaf; 9, node; 10, internode; 11, axillary bud; 12, lamina joint of the 30-d seedling. Bars = 2 mm. E, Relative expression levels of OsGRF7 in different tissues analyzed with RT-qPCR. OsUBI was used as an internal reference. Values are means ± sd of three biological replicates.

Figure 3.

ChIP analysis of the OsGRF7 target genes. A, Distribution of OsGRF7-binding peaks in the rice genome. B, Genes reproducibly associated with OsGRF7-binding sites in YP1 or YP2. C, Putative OsGRF7-binding motif predicted by DREME. D, Expression and purification of OsGRF7 protein in Escherichia coli. The red arrowhead indicates the purified OsGRF7 protein. E, EMSA validation of interaction between OsGRF7 and motif ACRGDA (ACAGTA). TAAGTA was used as the control. F, OsGRF7-binding peaks in the promoters of OsCYP714B1 and OsARF12. Blue boxes indicate probe locations. Bar = 1 kb. G, ChIP-qPCR validation of OsGRF7-binding sites in the promoters of OsCYP714B1 and OsARF12. The fold enrichment was normalized against the promoter of OsUBI. No addition of antibodies (NoAbs) served as a negative control. H and I, Relative expression levels of OsCYP714B1 and OsARF12 in the lamina joint of OsGRF7 transgenic lines were detected with RT-qPCR. OsUBI was used as an internal reference. J and K, OsGRF7 activates OsCYP714B1 and OsARF12 promoter-luciferase fusion constructs in transient transactivation assays. LUC/REN, Firefly luciferase-to-renilla luciferase ratio. In G to K, values are means ± sd of three biological replicates. Asterisks indicate significant difference by two-tailed Student’s t test (*P < 0.05; **P < 0.01; and ***P < 0.001). L and M, EMSA validation of binding between OsGRF7 and the promoters of OsCYP714B1 and OsARF12. Two-fold, 10-fold, and 100-fold unmodified probes were used as competitors. The presence (+) or absence (−) of components in the reaction mixture is indicated. Red triangles indicate the sites of ACRGDA motifs on the promoter region.

Figure 4.

OsGRF7 regulates the expression of multiple IAA/GA-related genes. A, Relative expression levels of auxin signaling-related and GA synthesis-related genes in the lamina joint of the OsGRF7 transgenic lines. OsUBI was used as an internal reference. WT, Wild type. B to J, GRF7-GFP mediated the ChIP-qPCR enrichment (relative to the promoter region of OsUBI) of ACRGDA-containing fragments (marked with arrows) from the promoters of auxin signaling-related genes (ARF3, ARF4, ARF8, ARF9, PIN1b, PIN1d, and PIN8) and GA synthesis-related genes (SLR1 and SLRL1). The promoter region of OsUBI was used as a control. K and L, OsGRF7 activates ARF3, ARF4, ARF8, ARF9, PIN1b, PIN1d, PIN8, SLR1, and SLRL1 promoter-luciferase fusion constructs in transient transactivation assays. In B to J and L, values are means ± sd of three biological replicates. Asterisks indicate significant difference by two-tailed Student’s t test (*P < 0.05; **P < 0.01; and ***P < 0.001).

Figure 5.

Endogenous phytohormone contents and exogenous phytohormone sensitivity test of OsGRF7 transgenic lines. A and B, Quantification of GA (A) and IAA (B) derivatives in OsGRF7 transgenic seedlings detected with liquid chromatography-tandem mass spectrometry. Values are means ± sd of three biological replicates. Asterisks indicate significant difference by two-tailed Student’s t test (*P < 0.05). F.W., Fresh weight; ICA, indole-3-carboxaldehyde; ME-IAA, methyl indole-3-acetate; NAA, 1-naphthylacetic acid; nd, not detected (below the detection limit); WT, wild type. C, Immunohistochemical observation of IAA at the lamina joint of OsGRF7 transgenic lines. Anti-IAA antibody and Alexa 488-conjugated goat anti-rabbit IgG antibody were used. Bars = 100 μm. D, Phenotypes of OsGRF7 transgenic seedlings treated by different concentrations of phytohormones. The same control was used for 0 μm IAA and GA. Bars = 2 cm. E, Effects of different concentrations of IAA on primary root length in the wild type, GRF7OE-1, and GRF7RNAi-1. Values are means ± sd (n = 15, three biological replicates and five plants per replicate). F, Effects of different concentrations of GA₃ on the second sheath length in wild-type, GRF7OE-1, and GRF7RNAi-1 seedlings. Values are means ± sd (n = 15, three biological replicates and five plants per replicate). In E and F, different letters denote significant differences (P < 0.05) from Duncan’s multiple range test.

Figure 6.

Proposed model for the regulation of rice plant architecture by OsGRF7. In GRF7OE plants, OsGRF7 coactivates with OsGIFs to up-regulate the expression of downstream hormone-related genes through binding to ACRGDA motifs. Then the synthesis of GA₄ is inhibited, and the synthesis of IAA is promoted. Finally, the GRF7OE plants display a semidwarf and compact plant architecture. WT, Wild type.