A rice WRKY gene encodes a transcriptional repressor of the gibberellin signaling pathway in aleurone cells

Abstract

The molecular mechanism by which GA regulates plant growth and development has been a subject of active research. Analyses of the rice (Oryza sativa) genomic sequences identified 77 WRKY genes, among which OsWRKY71 is highly expressed in aleurone cells. Transient expression of OsWRKY71 by particle bombardment specifically represses GA-induced Amy32b alpha-amylase promoter but not abscisic acid-induced HVA22 or HVA1 promoter activity in aleurone cells. Moreover, OsWRKY71 blocks the activation of the Amy32b promoter by the GA-inducible transcriptional activator OsGAMYB. Consistent with its role as a transcriptional repressor, OsWRKY71 is localized to nuclei of aleurone cells and binds specifically to functionally defined TGAC-containing W boxes of the Amy32b promoter in vitro. Mutation of the two W boxes prevents the binding of OsWRKY71 to the mutated promoter, and releases the suppression of the OsGAMYB-activated Amy32b expression by OsWRKY71, suggesting that OsWRKY71 blocks GA signaling by functionally interfering with OsGAMYB. Exogenous GA treatment decreases the steady-state mRNA level of OsWRKY71 and destabilizes the GFP:OsWRKY71 fusion protein. These findings suggest that OsWRKY71 encodes a transcriptional repressor of GA signaling in aleurone cells.

Figure 1.

RT-PCR analysis of OsWRKY gene expression in aleurone cells. Total RNA was isolated from rice aleurone layers treated with no hormone, 1 μm GA₃, 20 μm ABA, or 1 μm GA₃ plus 20 μm ABA for 4, 24, and 48 h, respectively. Genomic DNA was isolated from rice 10-d-old seedlings using the cetyl-trimethyl-ammonium bromide method. P, Positive control, with rice genomic DNA as the template; C, No hormone control; G, GA treatment; A, ABA treatment; M, GA + ABA treatment. Expected RT-PCR fragment of the intron-containing genomic sequence for OsWRKY71 is 1393 bp; the corresponding cDNA is 1047 bp in length. RAmy3E, a GA-inducible gene; RAB21, an ABA-inducible gene; OsActin1, a constitutively expressed actin gene.

Figure 2.

Protein sequence alignment of OsWRKY71, AfABF2, and AtWRKY40 and domain structure of OsWRKY71. A, Alignment of the rice, wild oat, and Arabidopsis WRKY proteins. The deduced amino acid sequences were aligned by using ClustalW. Identical residues are shaded in black and residues chemically similar in gray. The putative nuclear localization signal (KRIR) and WRKY amino acid residues are labeled with rectangles, and amino acid residues potentially interacting with zinc ligands are pointed to with arrows. B, The OsWRKY71 protein sequence was analyzed for protein domain structures. Only three identified domains, nuclear localization signal (NLS), WRKY, and the zinc finger motif (Zn-F), are shown.

Figure 3.

OsWRKY71 proteins are targeted to nuclei of aleurone cells. Aleurone cells were transformed with UBI-GFP (A and B) or UBI-GFP:OsWRKY71 (C and D). After incubation for 24 h, the aleurone cells were stained with SYTO17 for nuclear localization (B and D), followed by examination of GFP expression (A and C). Arrows in A and B point to the same cell; so do those in C and D. The bars represent 20 μm.

Figure 4.

OsWRKY71 represses GA induction of the Amy32b promoter activity in a dosage-dependent manner. A, Schematic diagrams of the reporter and effector constructs used in the cobombardment experiment. Rectangles in the effector construct represent the exons of OsWRKY71 gene; bars between the rectangles denote the introns of this gene; thick lines represent terminators. B, The reporter construct, Amy32b-GUS, and the internal construct, UBI-Luciferase, were cobombarded into barley aleurone cells either with (+) or without (−) effector construct UBI-OsWRKY71 by using the same molar ratio of effector and reporter constructs. GUS activity was normalized in every independent transformation relative to the luciferase activity. Bars indicate GUS activity ±se after 24 h of incubation of the bombarded aleurone cells with (+) or without (−) 1 μm GA₃. Data are means ± se of four replicates. C, The effector construct, UBI-OsWRKY71, was cobombarded into barley aleurone cells along with the reporter construct Amy32b-GUS and the internal control construct UBI-Luciferase. The amount of reporter and internal control plasmid DNA was always constant, whereas that of effector varied with respect to the reporter as shown in the x axis. 100% means the same molar ratio of effector and reporter DNA was used. GUS activity was normalized in every independent transformation relative to the luciferase activity. The line indicates GUS activity ±se after 24 h of incubation of the bombarded aleurone cells with 1 μm GA₃. Data are means ± se of four replicates.

Figure 5.

OsWRKY71 has little effect on the ABA signaling in aleurone cells. A, Schematic diagrams of the reporter and effector constructs used in the transient experiment. HVA22 and HVA1 are two ABA inducible genes in barley aleurone cells. B, The reporter construct HVA22-GUS and the internal construct UBI-Luciferase were cobombarded into barley aleurone cells either with (+) or without (−) effector construct UBI-OsWRKY71 by using the same molar ratio of effector and reporter constructs. GUS activity was normalized in every independent transformation relative to the luciferase activity. Bars indicate GUS activity ±se after 24 h of incubation of the bombarded aleurone cells with (+) or without (−) 20 μm ABA. Data are means ± se of four replicates. C, The reporter construct HVA1-GUS and the internal construct UBI-Luciferase were cobombarded into barley aleurone cells either with (+) or without (−) effector construct UBI-OsWRKY71 by using the same molar ratio of effector and reporter constructs. GUS activity was normalized in every independent transformation relative to the luciferase activity. Bars indicate GUS activity ±se after 24 h of incubation of the bombarded aleurone cells with (+) or without (−) 20 μm ABA. Data are means ± se of four replicates.

Figure 6.

OsWRKY71 blocks the transactivation of Amy32b by OsGAMYB. A, Schematic diagrams of the reporter and effector constructs used in the cobombardment experiment. OsGAMyb is from a rice cDNA clone. B, OsWRKY71 suppresses OsGAMYB-activated expression of Amy32b. The reporter construct (Amy32b-GUS), effector constructs (UBI-OsGAMyb and UBI-OsWRKY71), and the internal construct (UBI-Luciferase), at an equal molar ratio, were cobombarded into barley aleurone cells. Transformed aleurone cells were incubated for 24 h without GA₃. GUS activity was normalized in every independent transformation relative to the luciferase activity. Data are means ± se of four replicates.

Figure 7.

OsWRKY71 interferes with OsGAMYB transactivation of Amy32b via binding to the TGAC cores within the promoter region. A, Partial nucleotide sequence of the Amy32b promoter and its mutant versions used as competitors in electrophoretic mobility-shift assays. Amy32b, wild-type Amy32b promoter; mTGAC1, the Amy32b promoter with the first W box mutated; mTGAC2, the Amy32b promoter with the second W box mutated; mTGACs, the Amy32b promoter with both W boxes mutated; mPyr, the Amy32b promoter with the Pyr box mutated. The W boxes (TGAC cores) and Pyr box are underlined. The O2S element is indicated with a rectangle. The mutated nucleotides are in lowercase. B, Electrophoretic mobility-shift assay with recombinant OsWRKY71. A 61-bp fragment probe containing the W box and Pyr box of the Amy32b promoter and 0.5 μg of GST:OsWRKY71 protein were used in each binding reaction. GST indicates the control binding reaction with GST only. The molar excess of each competitor used (20- and 200-fold) is indicated above each lane. The arrow indicates the DNA-protein complex. C, Schematic diagrams of the reporter and effector constructs used in the cobombardment experiment. D, The reporter constructs Amy32b-GUS and Amy32b(mTGACs)-GUS and the internal construct UBI-Luciferase were cobombarded into barley aleurone cells either with or without effector constructs UBI-OsGAMyb and UBI-OsWRKY71 by using the same molar ratio of effector and reporter constructs. GUS activity was normalized in every independent transformation relative to the luciferase activity. Bars indicate GUS activity ±se after 24 h of incubation of the bombarded aleurone cells with or without 1 μm GA₃. Data are means ± se of four replicates. Control means no GA and effector genes were included.

Figure 8.

Exogenous GA₃ treatment promotes OsWRKY71 degradation in aleurone cells. Embryoless half-seeds were transformed with UBI-GFP or UBI-GFP:OsWRKY71. After incubation for 24 h, the aleurone layers were treated with water or 100 μm GA₃ for 12 h, followed by examination of GFP fluorescence under confocal microscope. A, C, and E, The green fluorescence from GFP control and GFP:OsWRKY71 fusion protein, respectively, before GA treatment. Other sections show the images of the same scopes after GA treatment (B and F) or water-treated control (D). The bars represent 100 μm.

Figure 9.

Signaling components on the GA response pathway leading to expression of the Amy32b α-amylase gene in aleurone cells. SLN1 is a repressor, which is destabilized by GA treatment; GAMYB is a GA-inducible transcriptional activator. OsWRKY71 functions as a transcriptional repressor interacting with GAMYB to repress the GA response; GA down-regulates the expression of the OsWRKY71 gene at transcriptional or posttranscriptional level and destabilizes OsWRKY71 protein.