Differentially expressed genes during the imbibition of dormant and after-ripened seeds - a reverse genetics approach

Abstract

Background: Seed dormancy, defined as the incapability of a viable seed to germinate under favourable conditions, is an important trait in nature and agriculture. Despite extensive research on dormancy and germination, many questions about the molecular mechanisms controlling these traits remain unanswered, likely due to its genetic complexity and the large environmental effects which are characteristic of these quantitative traits. To boost research towards revealing mechanisms in the control of seed dormancy and germination we depend on the identification of genes controlling those traits.

Methods: We used transcriptome analysis combined with a reverse genetics approach to identify genes that are prominent for dormancy maintenance and germination in imbibed seeds of Arabidopsis thaliana. Comparative transcriptomics analysis was employed on freshly harvested (dormant) and after-ripened (AR; non-dormant) 24-h imbibed seeds of four different DELAY OF GERMINATION near isogenic lines (DOGNILs) and the Landsberg erecta (Ler) wild type with varying levels of primary dormancy. T-DNA knock-out lines of the identified genes were phenotypically investigated for their effect on dormancy and AR.

Results: We identified conserved sets of 46 and 25 genes which displayed higher expression in seeds of all dormant and all after-ripened DOGNILs and Ler, respectively. Knock-out mutants in these genes showed dormancy and germination related phenotypes.

Conclusions: Most of the identified genes had not been implicated in seed dormancy or germination. This research will be useful to further decipher the molecular mechanisms by which these important ecological and commercial traits are regulated.

Keywords: Arabidopsis thaliana; Delay of germination; Knockout lines; Seed performance; Transcriptromics.

Fig. 1

Microarray analysis of dormant and after-ripened seeds after 24 h of imbibition of five genotypes with differing dormancy levels; Ler, NILDOG1, NILDOG2, NILDOG3 and NILDOG6. a After-ripening requirement of the five genotypes. On the right graphical representations of the NILs are depicted showing the 5 chromosomes with the introgressed regions (in red) in an otherwise Ler background (in green). b Venn diagrams showing the number of genes that are differentially expressed (P < 0.0001) in dormant (D-up) and after ripened (AR-up) 24-h imbibed seeds of different genotypes. For each genotype the total number of differential expressed genes is indicated between brackets. In the intersection of all genotypes the number of genes that are investigated in this study are presented, 46 and 25 for the D-up and AR-up set, respectively. c Heat map consisting of 245 NILDOG1 D-up genes (P < 0.0001). The significance of these genes in the other genotypes is indicated, the white color indicates the genes that are not significantly different in the other genotypes (P < 0.01). d Heat map consisting of 159 NILDOG1 AR-up genes (P < 0.0001). The significance of these genes in the other genotypes is indicated, the white color represents the genes that are not significantly different in the other genotypes (P < 0.01). e Germination behaviour of freshly harvested seeds of Ler, dog1 and NILDOG1. f Box plot showing the expression of the 45 D-up genes in freshly harvested imbibed Ler, dog1 and NILDOG1 seeds (expression data taken from Dekkers et al. [16]). g Box plot showing the expression of the 25 D-up genes in freshly harvested imbibed Ler, dog1 and NILDOG1 seeds

Fig. 2

Plant phenotypes of T-DNA knock-out lines in comparison with wild type Columbia (Col). a Aborted seeds in siliques from heterozygous T-DNA lines with insertions in FBA2 (AT1G06080) and ADS1 (AT4G38970). b Four-week old plants of the NAD(P)-BINDING ROSSMANN-FOLD SUPER FAMILY PROTEIN (nbrsfp; KO1, AT2G29300) and SUCCINATE-SEMIALDEHYDE DEHYDROGENASE mutant (ssadh1, KO25, At1G79440) (c) nbrsfp, Col and ssadh1 6 weeks after germination

Fig. 3

Germination behaviour of knock-out mutants (KO) in dormancy (left) and after –ripened (right) upregulated genes: (a) Average DSDS50 (Days of Seed Dry Storage until 50% germination) values. b germination after accelerated aging. c germination in salt 130 mM; d) in mannitol (−1 MPa) and e) in ABA(0.15 μM) solutions. Significant differences are indicated (*P < 0.05 and **P < 0.01). There are differences in Col-0 values between the different experiments, however, every knock-out line has been compared to the Col-0 that was grown in the same experiment