Arabidopsis thaliana DOF6 negatively affects germination in non-after-ripened seeds and interacts with TCP14

Abstract

Seed dormancy prevents seeds from germinating under environmental conditions unfavourable for plant growth and development and constitutes an evolutionary advantage. Dry storage, also known as after-ripening, gradually decreases seed dormancy by mechanisms not well understood. An Arabidopsis thaliana DOF transcription factor gene (DOF6) affecting seed germination has been characterized. The transcript levels of this gene accumulate in dry seeds and decay gradually during after-ripening and also upon seed imbibition. While constitutive over-expression of DOF6 produced aberrant growth and sterility in the plant, its over-expression induced upon seed imbibition triggered delayed germination, abscisic acid (ABA)-hypersensitive phenotypes and increased expression of the ABA biosynthetic gene ABA1 and ABA-related stress genes. Wild-type germination and gene expression were gradually restored during seed after-ripening, despite of DOF6-induced over-expression. DOF6 was found to interact in a yeast two-hybrid system and in planta with TCP14, a previously described positive regulator of seed germination. The expression of ABA1 and ABA-related stress genes was also enhanced in tcp14 knock-out mutants. Taken together, these results indicate that DOF6 negatively affects seed germination and opposes TCP14 function in the regulation of a specific set of ABA-related genes.

Fig. 1.

DOF6 expression patterns. DOF6 mRNA levels relative to UBC21 in: (A) wild-type plants in different plant organs (L, adult rosette leaves; F, flowers; R, roots; S, pool of developing siliques from 4 to 15 days after pollination (dap); DS, dry seeds); (B) siliques at different dap and (C) seeds at different hours after imbibition (hai). Means and standard errors for three replicates are shown. (D) Expression of the GUS reporter gene driven by the DOF6 promoter in seeds imbibed for 3 h. Seed covers were removed before incubating with the staining solution. (E, F) In situ hybridization of DOF6 transcripts in Col-0 seed sections. Seeds were imbibed for 24 h and hybridized using antisense (E) and sense (F) DOF6 probes. Gene-specific mRNA levels are relative to UBC21. Bars, 100 μm (D), 100 and 50 μm (E and F).

Fig. 2.

DOF6 over-expression produces growth and developmental defects on the plant. (A) DOF6 mRNA levels in rosette leaves of wild-type (WT) and p35S::DOF6 transgenic plants. Representative independent transgenic lines are shown. (B and C) Three- and eight-week-old plants over-expressing DOF6 (p35S::DOF6), respectively and show a dwarf phenotype compared to the WT and are unable to produce seeds. (D) DOF6 mRNA levels in WT and two representative ioexDOF6 lines, before and 16 h after spraying with 50 μM estradiol. (E and F) WT and two representative ioexDOF6 lines germinated on half-strength Murashige and Skoog agar with 50 μM estradiol added every 2 days (E) and without estradiol (F); pictures were taken 3 weeks after sowing. Gene-specific mRNA levels are relative to UBC21.

Fig. 3.

Germination of ioexDOF6 seeds and DOF6 expression in freshly harvested (FH) and after-ripened (AR) seeds. (A) DOF6 expression levels in FH wild type (WT) and two representative ioexDOF6 lines upon imbibition in 50 μM estradiol. (B and C) Germination of FH WT and ioexDOF6 seeds imbibed in 50 μM estradiol (C) and controls without estradiol (B). (D) Germination of AR WT and ioexDOF6 seeds in 50 μM estradiol. (E) Germination percentage of WT and ioexDOF6 seeds at 72 hours after imbibition (hai) in 50 μM estradiol after different weeks of dry storage. (F) DOF6 mRNA levels in FH and in AR WT dry seeds and 24 hai. Gene-specific mRNA levels are relative to UBC21.

Fig. 4.

ABA effect on DOF6 expression and ioexDOF6 seed germination. (A and B) DOF6 expression by quantitative RT-PCR in freshly harvested (FH; A) and after-ripened (AR; B) Col-0 seeds imbibed in water with 2 and 5 μM ABA, respectively. (C and D) Germination of FH (C) and AR (D) wild-type (WT) and ioexDOF6 seeds in water supplemented with 50 μM estradiol and 0.5 and 1 μM ABA, respectively. Germinating percentages are represented as the mean±standard error from three replicates. Germination patterns were confirmed in two different seed batches. Gene-specific mRNA levels are relative to UBC21.

Fig. 5.

mRNA levels of ABA-related genes in germinating freshly harvested (FH) ioexDOF6 seeds. Expression of genes involved in ABA metabolism and signalling (A) and ABA-inducible genes (B) was compared by quantitative RT-PCR between wild-type (WT) and ioexDOF6 FH seeds 48 hours after imbibition in 50 μM estradiol. Gene-specific mRNA levels are relative to UBC21. All the UBC21 normalized expression values are relative to the values obtained for each gene in the WT sample.

Fig. 6.

DOF6 interaction with TCP14. (A) Diploid cells carrying BD-DOF6 and AD-TCP14 (TCP14) or BD-DOF6 and AD empty vector (control –) were grown on the appropriate selection media with increasing concentrations of 3-amino-1,2,4-triazole (3-AT). (B) Quantification of the DOF6–TCP14 interaction in yeast using the β-galactosidase reporter gene. An AD-TCP1 construct was used as a negative control. (C) Bimolecular fluorescent complementation assays in onion epidermal cells by particle bombardment. TCP14 and DOF6 were fused to the C- and N-terminal fragments, respectively, of yellow fluorescent protein (YFP) and co-bombarded over epidermal onion cells. Images were obtained with a fluorescence microscope.

Fig. 7.

mRNA levels of DOF6 and ABA-related genes in germinating tcp14 mutant seeds. (A) Expression of TCP14, ABA1, sHSP17.4, and sHSP22 was compared by quantitative RT-PCR between freshly harvested wild-type (WT) and tcp14 imbibed seeds. WT expression levels are 1 in all cases. (B) mRNA levels of DOF6 and TCP14 were quantified in tcp14 and ioexDOF6 FH seeds, respectively. WT and ioexDOF6 seeds used in the left panel were imbibed for 24 h in 50 μM estradiol. Gene-specific mRNA levels are relative to UBC21.