Single seeds exhibit transcriptional heterogeneity during secondary dormancy induction

Seeds are highly resilient to the external environment, which allow plants to persist in unpredictable and unfavorable conditions. Some plant species have adopted a bet-hedging strategy to germinate a variable fraction of seeds in any given condition, and this could be explained by population-based threshold models. Here, in the model plant Arabidopsis (Arabidopsis thaliana) we induced secondary dormancy to address the transcriptional heterogeneity among seeds that leads to binary germination/non-germination outcomes. We developed a single seed RNA-seq strategy that allowed us to observe a reduction in seed transcriptional heterogeneity as seeds enter stress conditions, followed by an increase during recovery. We identified groups of genes whose expression showed a specific pattern through a time course and used these groups to position the individual seeds along the transcriptional gradient of germination competence. In agreement, transcriptomes of dormancy-deficient seeds (mutant of DELAY OF GERMINATION 1 gene) showed a shift towards higher values of the germination competence index. Interestingly, a significant fraction of genes with variable expression encoded translation-related factors. In summary, interrogating hundreds of single seed transcriptomes during secondary dormancy-inducing treatment revealed variability among the transcriptomes that could result from the distribution of population-based sensitivity thresholds. Our results also showed that single seed RNA-seq is the method of choice for analyzing seed bet-hedging-related phenomena. Overall design: 1) Three libraries, each consisting of 32 seeds were prepared for each time point of secondary dormancy induction time course for Arabidopsis seeds (1h, 1d, 3d, 5d, 7d, 7d+24h, pool-split control). 2) Three libraries, each consisting of 32 seeds were prepared for two time points of secondary dormancy induction (3d, 7d+24h) and two genotypes (Col-0 and dog1-4 mutant). 3) Comparison of dry seed transcriptome of Col-0 and dog1-4 mutant (three biological replicas).