Sequence Polymorphisms at the REDUCED DORMANCY5 Pseudophosphatase Underlie Natural Variation in Arabidopsis Dormancy

Abstract

Seed dormancy controls the timing of germination, which regulates the adaptation of plants to their environment and influences agricultural production. The time of germination is under strong natural selection and shows variation within species due to local adaptation. The identification of genes underlying dormancy quantitative trait loci is a major scientific challenge, which is relevant for agricultural and ecological goals. In this study, we describe the identification of the DELAY OF GERMINATION18 (DOG18) quantitative trait locus, which was identified as a factor in natural variation for seed dormancy in Arabidopsis (Arabidopsis thaliana). DOG18 encodes a member of the clade A of the type 2C protein phosphatases family, which we previously identified as the REDUCED DORMANCY5 (RDO5) gene. DOG18/RDO5 shows a relatively high frequency of loss-of-function alleles in natural accessions restricted to northwestern Europe. The loss of dormancy in these loss-of-function alleles can be compensated for by genetic factors like DOG1 and DOG6, and by environmental factors such as low temperature. RDO5 does not have detectable phosphatase activity. Analysis of the phosphoproteome in dry and imbibed seeds revealed a general decrease in protein phosphorylation during seed imbibition that is enhanced in the rdo5 mutant. We conclude that RDO5 acts as a pseudophosphatase that inhibits dephosphorylation during seed imbibition.

Figure 1.

Complementation of DOG18 by RDO5. A, Germination after different periods of dry storage of seeds from Ler, NIL DOG18, and six independent transgenic NIL DOG18 lines containing the RDO5 gene from Ler. Shown are means ± se of six to eight independent batches of seeds for each genotype. B, Gene structure of RDO5 and natural polymorphisms identified in the An-1, Fei-0, and Kas-2 accessions compared to Ler. Exons are shown as black boxes and introns as lines. C, Immunoblot analysis of RDO5:HA protein accumulation in seeds of the six NIL DOG18 complementation transgenic lines from A. Coomassie Brilliant Blue staining was used as a loading control.

Figure 2.

Complementation of An-1 and Cam61 by RDO5. A and B, Germination after different periods of dry storage of seeds from transgenic lines containing the RDO5 Ler allele in An-1 (A) or Cam61 (B) background. Shown are averages ± se of six to eight independent batches of seeds for each genotype. C and D, qRT-PCR analysis of APUM9 transcript levels in dry and 6HAI seeds in An-1 (C), Cam61 (D), and their transgenic lines. The expression values were normalized using ACT8 as control. n = 3 biological replicates; error bars represent se.

Figure 3.

Natural loss-of-function mutations in the DOG18 gene. Natural mutations causing predicted DOG18 loss-of-function alleles are divided into five groups that are shown in separate rows. Location of mutations for the individual accessions are indicated. Exons (black boxes) are connected with horizontal lines representing intronic regions of DOG18. The location of the predicted PP2C domain is indicated at the top.

Figure 4.

Geographic distribution, population structure, and haplotype network of accessions containing RDO5 loss-of-function alleles. A, Geographical distribution of 42 accessions harboring RDO5 predicted loss-of-function alleles. B, Population structure of 95 accessions containing functional or predicted loss-of-function RDO5 alleles originating from Sweden, the UK, and western Europe at K = 3. Black stars indicate accessions carrying RDO5 loss-of-function alleles. C, RDO5 haplotype network. Haplotypes are represented by circles with sizes proportional to the number of populations containing that haplotype. Each node represents a single mutation. Black stars indicate accessions or groups carrying RDO5 loss-of-function alleles.

Figure 5.

Genetic modifiers of the RDO5 phenotype. A, Dormancy level of 11 accessions with predicted loss-of-function RDO5 alleles. Col and Cvi are used as low- and high-dormancy controls. POG-0, HR-10, NFA-8, Vind-1, and NFA-10 belong to the nonsense mutation group; An-1, Fei-0, Lac-3, Cam61, and Durh-1 belong to the Indels group; and Fr-2 belongs to the gene-loss group. Shown are means ± se of six to eight independent batches of seeds for each genotype. DSDS50, days of seed dry storage required to reach 50% germination. B, Immunoblot analysis of DOG1 protein accumulation using a DOG1 antibody in the 11 accessions with predicted loss-of-function RDO5 alleles. A band from Coomassie Brilliant Blue staining was used as a loading control. The bottom panel indicates the DOG1 haplotype of the accessions, where ECCY is a strong allele and DSY is a weak allele (Nakabayashi et al., 2015). C, The correlation of dormancy level and DOG1 protein abundance (quantified from B and normalized by the loading control). D, A strong DOG6 allele from the Shahdara accession enhances dormancy of the rdo5-1 mutant (in Ler background). Germination percentages were determined in freshly harvested seeds. Shown are means ± se of six to eight independent batches of seeds for each genotype. **P < 0.01. E, qRT-PCR analysis of APUM9 transcript levels in Ler, rdo5-1 (Ler background), NIL DOG6, and rdo5-1 NIL DOG6 dry and 6HAI seeds. Inset, APUM9 expression in dry seeds from the different genotypes at a magnified scale. The expression values were normalized using ACT8 as control. n = 3 biological replicates; error bars represent se.

Figure 6.

Maternal temperature affects the RDO5 dormancy phenotype. A, Germination of NIL DOG1, rdo5-1, dog1-1, and rdo5-1 dog1-1 freshly harvested seeds that matured under a d/n regime of 22/16°C or 16/14°C. Shown are means ± se of six to eight independent batches of seeds for each genotype. B, qRT-PCR analysis of RDO5 transcript levels in NIL DOG1 dry and 6HAI seeds that matured under a d/n regime of 22/16°C or 16/14°C. The expression values were normalized using ACT8 as control. n = 3 biological replicates; error bars represent se. C, qRT-PCR analysis of APUM9 transcript levels in NIL DOG1, rdo5-1, dog1-1, and rdo5-1 dog1-1 dry and 6HAI seeds that matured under a d/n regime of 22/16°C or 16/14°C. The expression values were normalized using ACT8 as control. n = 3 biological replicates; error bars represent se.

Figure 7.

RDO5 functions as a pseudophosphatase. A, Phosphatase activity is restored in RDO5 after back-mutations. Phosphatase activity of RDO5 and RDO5 back-mutation (RDO5bm) proteins was measured in vitro using the RRA(phosphoT)VA peptide as a substrate. ΔC-terN88AHG3 was used as a positive control. Data are averages ± se from three replicates. B, Venn diagram analyses showing common and differential distribution of phosphorylated sites identified in NIL DOG1 and rdo5-1 after 6 h imbibition compared with dry seeds. C to E, Localization in the SeedNet network of differentially phosphorylated proteins from wild-type NIL DOG1 (C), rdo5-1 (D), and differentially phosphorylated proteins present in rdo5-1 but not in NIL DOG1 (E) after 6 h imbibition compared with dry seeds. The regions outlined in green correspond to clusters associated with dormancy (region 1) or germination (regions 2 and 3). The red dots represent proteins with decreased phosphorylation levels and the blue dots represent proteins with increased phosphorylation levels.