Arabidopsis WRKY6 Transcription Factor Acts as a Positive Regulator of Abscisic Acid Signaling during Seed Germination and Early Seedling Development

Abstract

The phytohormone abscisic acid (ABA) plays important roles during seed germination and early seedling development. Here, we characterized the function of the Arabidopsis WRKY6 transcription factor in ABA signaling. The transcript of WRKY6 was repressed during seed germination and early seedling development, and induced by exogenous ABA. The wrky6-1 and wrky6-2 mutants were ABA insensitive, whereas WRKY6-overexpressing lines showed ABA-hypersensitive phenotypes during seed germination and early seedling development. The expression of RAV1 was suppressed in the WRKY6-overexpressing lines and elevated in the wrky6 mutants, and the expression of ABI3, ABI4, and ABI5, which was directly down-regulated by RAV1, was enhanced in the WRKY6-overexpressing lines and repressed in the wrky6 mutants. Electrophoretic mobility shift and chromatin immunoprecipitation assays showed that WRKY6 could bind to the RAV1 promoter in vitro and in vivo. Overexpression of RAV1 in WRKY6-overexpressing lines abolished their ABA-hypersensitive phenotypes, and the rav1 wrky6-2 double mutant showed an ABA-hypersensitive phenotype, similar to rav1 mutant. Together, the results demonstrated that the Arabidopsis WRKY6 transcription factor played important roles in ABA signaling by directly down-regulating RAV1 expression.

Fig 1. ABA-sensitivity of wrky6 mutants and WRKY6-overexpressing lines.

A, Expression of WRKY6 was analyzed by qRT-PCR in wild-type plants (WT) during seed germination and early seedling development. The wild-type imbibed seeds were germinated and grown on MS medium, and then the plants were harvested at the indicated time. Data are shown as mean ± SE (n = 3). B, qRT-PCR analysis of WRKY6 expression in response to exogenous ABA. Wild-type imbibed seeds were germinated on MS medium (MS) or MS medium with 0.5 M ABA (MSABA) for 1 d, and then the seeds were harvested. Data are shown as mean ± SE (n = 3). C, qRT-PCR analysis of WRKY6 expression in 7-d-old wild-type seedlings treated with or without 100 M ABA for 3 h. Data are shown as mean ± SE (n = 3). D, Expression of WRKY6 was analyzed by qRT-PCR in the wrky6 mutants (wrky6-1 and wrky6-2) and WRKY6-overexpressing lines (35S:WRKY6-5 and 35S:WRKY6-9). Data are shown as mean ± SE (n = 3). E, Phenotypic comparison. Imbibed seeds were transferred to MS or MS 0.5 μM ABA medium and grown for 10 d. F-H, Seed germination assay. Imbibed seeds were transferred to MS (F), MS medium containing 0.5 M ABA (G) or 2 M ABA (H), and then the seed germination rates were calculated at the indicated time. Data are shown as mean ± SE (n = 3). More than 300 seeds were measured in each replicate. I, Cotyledon-greening analysis. Imbibed seeds were transferred to MS or MS 0.5 μM ABA medium for 7 d before determining cotyledon-greening percentages. Data are shown as mean ± SE (n = 3). More than 300 seeds were measured in each replicate. J-K, Primary root length measurement with and without ABA addition. The 4-d-old seedlings were transferred to MS or MS 15 μM ABA medium for 7 d, and then the photos were taken and the primary root length was measured. Asterisks in G, H, I and K indicate statistically significant differences compared with wild-type plants: *, P 0.05; **, P 0.01. Wild-type plant (WT) was used as a control (#).

Fig 2. Expression of ABA-responsive genes in wrky6 mutants and WRKY6-overexpressing lines.

The imbibed seeds were germinated and grown on MS medium for 7 d, and then the seedlings were harvested for qRT-PCR. Data are shown as mean ± SE (n = 3). Asterisks indicate statistically significant differences compared with wild-type plants: *, P 0.05; **, P 0.01. Wild-type plant (WT) was used as a control (#).

Fig 3. Expression of ABA-responsive genes in wrky6 mutants and wild-type seedlings treated with exogenous ABA.

The 7-d-old wrky6 mutants and wild-type seedlings were transferred to MS solution with or without 100 μM ABA for 3 h, and then the seedlings were harvested for qRT-PCR. Data are shown as mean ± SE (n = 3). Asterisks indicate statistically significant differences compared with relevant wild-type plants (WT): *, P 0.05; **, P 0.01.

Fig 4. Expression of WRKY6, RAV1 and ABIs in wild-type plants during seed germination and early seedling development.

The imbibed wild-type seeds were transferred to the MS medium with or without 0.5 μM ABA, and then the plants were harvested at the indicated time for qRT-PCR. Data are shown as mean ± SE (n = 3). Asterisks indicate statistically significant differences compared with relevant wild-type plants (WT): *, P 0.05; **, P 0.01.

Fig 5. WRKY6 directly represses RAV1 expression.

A, Schematic representation of RAV1 locus. RAV1 putative promoter is indicated by black line showing relative positions of W-box motifs (gray lines), and transcribed sequence by black box (exon) and gray boxes (untranslated regions). Relative positions and sizes of different PCR-amplified fragments are indicated by black lines under the W-boxes. The sequence of W-box is shown in blue and the TATA box is shown in red. B, Transient overexpression of WRKY6 fused to ProRAV1:GUS in Nicotiana benthamiana leaves. Data are shown as mean ± SE (n = 4). Asterisks indicate statistically significant differences: **, P 0.01. C, EMSA of WRKY6 binding to RAV1 promoter in vitro. Each biotin-labeled DNA probe was incubated with WRKY6-His protein. The mutation probes of P1 and P2 have the mutated W-box (TTGACC was replaced by TACGTC). D, Immunoblot analysis of WRKY6 protein. The 7-d-old wrky6-2 mutant and wild-type seedlings were transferred to MS solution with or without 100 μM ABA for 3 h, and then the seedlings were harvested for immunoblot analysis using anti-WRKY6 antibody. The relative band intensities of WRKY6, normalized relative to the intensity with the value of ACTIN (as 100%), are indicated by numbers below the bands. E, ChIP-qPCR assay of WRKY6 binding to RAV1 promoter in vivo. The 7-d-old wild-type seedlings were transferred to MS solution with or without 100 μM ABA for 3 h, and then the seedlings were harvested for ChIP-qPCR assay using anti-WRKY6 antibody. Data are shown as mean ± SE (n = 3). Asterisks indicate statistically significant differences: **, P 0.01.

Fig 6. Overexpression of RAV1 impairs the ABA-sensitive phenotypes of WRKY6-overexpressing line.

A, The expression of WRKY6 and RAV1 was tested by qRT-PCR in 35S:WRKY6-9, RAV1 OE2, 35S:WRKY6-9/RAV1 OE2 and wild-type plants (WT). Data are shown as mean ± SE (n = 3). B, Phenotypic comparison. Imbibed seeds were germinated and grown on MS medium (MS) or MS medium containing 0.5 μM ABA (MS + 0.5 μM ABA) for 10 d. C-D, Seed germination assay. Imbibed seeds were transferred to MS medium (C) or MS + 0.5 μM ABA medium (D), and then the seed germination rates were calculated at the indicated time. Data are shown as mean ± SE (n = 3). More than 300 seeds were measured in each replicate. E, Cotyledon-greening analysis. Imbibed seeds were germinated and grown on MS or MS + 0.5 μM ABA medium for 7 d before determining cotyledon-greening percentage. Data are shown as mean ± SE (n = 3). More than 300 seeds were measured in each replicate. Asterisks in D and E indicate statistically significant differences compared with wild-type plants: *, P < 0.05; **, P < 0.01. Wild-type plant (WT) was used as a control (#).

Fig 7. Expression of ABI3, ABI4 and ABI5 in 35S:WRKY6-9, RAV1 OE2, 35S:WRKY6-9/RAV1 OE2 and wild-type plants.

The imbibed seeds were germinated and grown on MS medium for 5 d, and then the seedlings were harvested for qRT-PCR. Data are shown as mean ± SE (n = 3). Asterisks indicate statistically significant differences compared with wild-type plants: *, P 0.05; **, P 0.01. Wild-type plant (WT) was used as a control (#).

Fig 8. Expression of ABFs, Ems and SnRK2s in the wrky6-2 mutant, RAV1 OE2, wrky6-2 RAV1 OE transgenic lines and wild-type plants.

The genes expression was tested by qRT-PCR in the wrky6-2 mutant, RAV1 OE2, wrky6-2 RAV1 OE transgenic lines (T1) and wild-type plants (WT). Three technical replicates were performed. Asterisks indicate statistically significant differences compared with wild-type plants: *, P 0.05; **, P 0.01. Wild-type plant (WT) was used as a control (#).

Fig 9. The ABA-insensitivity of the wrky6 mutant is abolished by suppression of RAV1.

A, The transcript levels of WRKY6 and RAV1 were tested by qRT-PCR. Data are shown as mean ± SE (n = 3). B, Phenotypic comparison. Imbibed seeds were transferred to MS medium (MS) or MS medium containing 0.5 μM ABA (MS + 0.5 μM ABA) for 10 d. C, Cotyledon-greening analysis. Imbibed seeds were transferred to MS or MS + 0.5 μM ABA medium for 7 d before determining cotyledon-greening percentage. Data are shown as mean ± SE (n = 3). More than 300 seeds were measured in each replicate. D, Expression of ABI3, ABI4, and ABI5 was tested by qRT-PCR in the wrky6-2 mutant, RAV1-U, wrky6-2 RAV1-U double mutant and wild-type plants. Each data represents the mean ± SE (n = 3). Asterisks indicate statistically significant differences compared with wild-type plants: *, P < 0.05; **, P < 0.01. Wild-type plants (WT) were used as a control (#).

Fig 10. Disruption of RAV1 abolishes the ABA-insensitivity of the wrky6 mutant.

A, Diagram of RAV1 showing two target sites (C1 and C2) for CRISPR/Cas9 technology. PAM motifs are marked with bold letters. The coding sequence (CDS) and untranslated regions (UTR) of RAV1 are indicated by black box and gray boxes, separately. B, The rav1 mutant and rav1 wrky6-2 double mutant were generated by CRISPR/Cas9 technology. The mutation in the RAV1 gene is evaluated by sequencing, and the mutant sites in RAV1 are indicated by red letters and arrows. C, qRT-PCR analysis of WRKY6 expression in the wrky6-2 mutant, rav1 mutant, rav1 wrky6-2 double mutant and wild-type plants (WT). Data are shown as mean ± SE (n = 3). D, Phenotypic comparison. Imbibed seeds were transferred to MS or MS + 0.5 μM ABA medium for 10 d. E, Cotyledon-greening analysis. Imbibed seeds were transferred to MS or MS + 0.5 μM ABA medium for 7 d before determining cotyledon-greening percentage. Data are shown as mean ± SE (n = 3). F, qRT-PCR analysis of ABIs in the wrky6-2 mutant, rav1 mutant, rav1 wrky6-2 double mutant and wild-type plants (WT) treated with or without exogenous ABA treatment. The 7-d-old seedlings were transferred to MS solution with or without 100 μM ABA for 3 h, and then the seedlings were harvested for qRT-PCR. Data are shown as mean ± SE (n = 3). Asterisks indicate statistically significant differences compared with relevant wild-type plants (WT): *, P 0.05; **, P 0.01.

Fig 11. WRKY6 can not directly regulate the expression of ABI3, ABI4 and ABI5.

A, Diagrams of ABI3, ABI4 and ABI5 promoters showing relative positions of W-box motifs (gray lines). Relative positions and sizes of different PCR-amplified fragments are indicated by black lines under the W boxes. The gray bent lines indicate the introns within the 5’UTR of ABI3 and ABI5. B-D, EMSA of WRKY6 binding to promoters of ABI3, ABI4 and ABI5 in vitro. Each biotin-labeled DNA probe was incubated with WRKY6-His protein. The mutation probes have the mutated W-box (TTGACC was replaced by TACGTC). E, Transient overexpression of WRKY6 fused to ProABIs:GUS in Nicotiana benthamiana leaves. Data are shown as mean ± SE (n = 6).

Fig 12. Hypothetical model of WRKY6/RAV1/ABIs-regulatory pathway in plant responses to ABA signaling during seed germination and early seedling development.

ABA induces the activity of WRKY6, and WRKY6 binds to the RAV1 promoter to repress RAV1 expression. RAV1 directly represses the expression of ABI3, ABI4 and ABI5, which promote seed germination and early seedling development.