Mutations in the Dof zinc finger genes DAG2 and DAG1 influence with opposite effects the germination of Arabidopsis seeds

Abstract

We describe the Arabidopsis gene DAG2 encoding a Dof zinc finger protein and show that it is involved in the control of seed germination. An Arabidopsis mutant line with a T-DNA insertion in DAG2 isolated by reverse genetics produces seeds that are substantially more dependent than the wild type on the physical stimuli-light and cold treatment-that promote germination. Mutant dag2 seeds also are less sensitive to the germination-promotive effect of gibberellins, because a 10-fold higher amount of gibberellins is needed to restore germination when endogenous gibberellin biosynthesis is blocked. The seed germination characteristics of the dag2 mutant are opposite to those of dag1, a knockout mutant of another Dof gene (DAG1) that we showed previously to be involved in the control of seed germination, and are similar to those of plants that overexpress DAG1. The promoter of the DAG2 gene is active specifically in the vascular system of the mother plant but not in the embryo, and segregation analysis indicates that the effect of the dag2 mutation is maternal. Both characteristics are in common with DAG1; additionally, the DAG1 and DAG2 proteins share high sequence homology and an identical zinc finger domain. These data suggest, and the germination phenotype of the double mutant is compatible with, a model whereby the zinc finger proteins DAG1 and DAG2 act on a maternal switch that controls seed germination, possibly by regulating the same gene(s).

Figure 1.

DAG2 and DAG1 Proteins Are Very Closely Related and Share an Identical DNA Binding Domain. Alignment of the amino acid sequences of the proteins encoded by the Dof genes DAG2 and DAG1. Green indicates identical amino acid residues, and red indicates conservative substitutions. The Dof domain is underlined.

Figure 2.

DAG1 and DAG2 Genes Have Similar Structures. Insertion of T-DNA results in a truncated DAG2-GUS fusion (Δ-DAG2::GUS) with a very low level of expression in the dag2 mutant. (A) Structure of the DAG2 and DAG1 genes and position of the T-DNA insert in the dag2 mutant. The T-DNA is not drawn to scale. Black boxes indicate exons, and dashed boxes indicate Dof domains. RB and LB, T-DNA right and left border, respectively. Numbers indicate nucleotide positions. (B) Structure of the DAG2 transcript in the dag2 mutant. RNA gel blot hybridization of the DAG2 5′ region upstream of the Dof domain (nucleotides 1 to 220) with total RNA extracted from leaves of Ws and dag2 plants. ATL18 is the mRNA of the Arabidopsis ribosomal protein L18 used to normalize the blots.

Figure 3.

Mutant dag2 and dag1 Plants Are Taller and Shorter, Respectively, Than Arabidopsis Ws Plants. The dag2 plant is at left, the Ws plant is in the center, and the dag1 plant is at right.

Figure 4.

Germination of Mutant dag2 Seeds Is More Dependent on Light and Stratification Than Germination of Wild-Type Seeds, Whereas Germination of Mutant dag1 Seeds Is Less Dependent on These Factors. (A) Germination rates under standard illumination conditions with seeds pretreated at 4°C. Closed circles, dag2 seeds; closed triangles, Ws seeds; closed squares, dag1 seeds. (B) Germination in the dark with seeds pretreated at 4°C. The histogram reports germination percentages scored on day 5. (C) Germination rates under standard illumination conditions with seeds not pretreated at 4°C. Closed circles, dag2 seeds; closed triangles, Ws seeds; closed squares, dag1 seeds. (D) Germination in the dark with seeds not pretreated at 4°C. The histogram reports germination percentages scored on day 5.

Figure 5.

Mutant dag2 and dag1 Seeds Require Much Higher and Much Lower Concentrations of GAs, Respectively, Than Wild-Type Seeds to Germinate. Both mutant seeds were as sensitive to abscisic acid as wild-type seeds. Germination of seeds pretreated at 4°C was under standard illumination conditions. Closed circles, dag2 seeds; closed triangles, Ws seeds; closed squares, dag1 seeds. (A) Germination percentages scored on day 5 in the presence of increasing concentrations of paclobutrazol. (B) Germination percentages scored on day 5 in the presence of 100 μM paclobutrazol and increasing concentrations of GAs (GA₄₊₇). (C) Germination percentages scored on day 5 in the presence of increasing concentrations of abscisic acid (ABA).

Figure 6.

Seeds from Plants Overexpressing DAG1 (35S::DAG1) Have a Lower Germination Rate Than Wild-Type Seeds. Germination of seeds not pretreated at 4°C was under standard illumination conditions. Closed triangles, Columbia ecotype seeds; open squares, 35S::DAG1 seeds.

Figure 7.

Promoters of the DAG2 and DAG1 Genes Show Very Similar Tissue and Developmental Specificities. Histochemical analysis of T3 Arabidopsis Ws plants harboring either the DAG2::GUS or the DAG1::GUS construct. (A), (C), (E), (G), (I), and (K) Activity of the DAG2::GUS construct in siliques, mature embryo, 7-day-old plantlet, cotyledon, leaf, and primary root, respectively. (B), (D), (F), (H), (J), and (L) Activity of the DAG1::GUS construct in siliques, mature embryo, 7-day-old plantlet, cotyledon, leaf, and primary root, respectively.

Figure 8.

The Effect of the Mutation of DAG2 Is Maternal. Germination rates under standard illumination conditions of seeds not pretreated at 4°C. Closed triangles, Ws seeds; open triangles, Ws × dag2 seeds; open circles, dag2 × Ws seeds.

Figure 9.

DAG1 Is Epistatic over DAG2. Germination rates under standard illumination conditions of seeds not pretreated at 4°C. Closed triangles, Ws seeds; closed diamonds, dag2 dag1 seeds.