SeedTransNet: a directional translational network revealing regulatory patterns during seed maturation and germination

Abstract

Seed maturation is the developmental process that prepares the embryo for the desiccated waiting period before germination. It is associated with a series of physiological changes leading to the establishment of seed dormancy, seed longevity, and desiccation tolerance. We studied translational changes during seed maturation and observed a gradual reduction in global translation during seed maturation. Transcriptome and translatome profiling revealed specific reduction in the translation of thousands of genes. By including previously published data on germination and seedling establishment, a regulatory network based on polysome occupancy data was constructed: SeedTransNet. Network analysis predicted translational regulatory pathways involving hundreds of genes with distinct functions. The network identified specific transcript sequence features suggesting separate translational regulatory circuits. The network revealed several seed maturation-associated genes as central nodes, and this was confirmed by specific seed phenotypes of the respective mutants. One of the regulators identified, an AWPM19 family protein, PM19-Like1 (PM19L1), was shown to regulate seed dormancy and longevity. This putative RNA-binding protein also affects the translational regulation of its target mRNA, as identified by SeedTransNet. Our data show the usefulness of SeedTransNet in identifying regulatory pathways during seed phase transitions.

Keywords: Arabidopsis thaliana; mRNA regulation; ribosome; seed germination; seed maturation; translatome profiling.

Fig. 1.

Ribosome and polysome abundance change during seed maturation. (A) Ribosome profiles from four developmental stages during seed maturation, 12, 15, 18, and 20 DAF (days after flowering). The profile is based on ribosome loading from equal seed dry weights. Approximate positions of ribosomal complexes are indicated. (B) Relative ribosome abundance at four developmental stages during seed maturation as determined by area under the curve in (A). Bars represent averages (n=3), error bars indicate ±SD, and the letters above each bar indicate the significance (Student’s t-test, P-value <0.05). (C) Comparison of ribosome profile of abi3-5 mutant and wild-type seeds. Both seed types were harvested at the same time when wild-type seeds were fully matured. The profile is based on ribosome loading from equal seed dry weights. (D) Relative ribosome content of wild-type and abi3-5 seeds as determined by the total area under the curve in the polysome and monosome regions of the density gradient in (C). Bars represent averages (n=3), error bars indicate ±SD, and asterisks indicate the significance (Student’s t-test, P-value <0.01).

Fig. 2.

Translational shift during Arabidopsis seed maturation. (A) The number of genes showing changed polysome occupancy (PO) during seed maturation. Seeds were harvested 12, 15, 18, and 20 days after flowering (DAF). The increase or decrease in PO is indicated by red and blue bars, respectively, with gene numbers above the bars. Genes were scored as changing if the PO change compared with the preceding time point was >2-fold and associated with a corrected P-value of <0.05. (B) PCA of normalized expression levels for the total RNA (T) and polysomal RNA (P) during seed maturation. Individual biological replicates are highlighted in the same colour. The total RNA and polysomal RNA samples are indicated as circles and squares, respectively. The different colours represent different time points during seed maturation. The first two components, PC1 and PC2, explain, respectively, 64.4% and 11.2% of the total variation. (C) Relationship between normalized abundance of genes in the polysomal and total RNA preparations. Genes associated with significantly changed PO between 12 and 15 DAF (MTS genes) are indicated in red (increased PO) or blue (decreased PO).

Fig. 3.

Dynamics of polysome occupancy (PO) during seed maturation and germination. The data used for the clustering analysis were combined from data presented in this study and the data from Bai et al. (2017). (A) Heatmap and hierarchical clustering of PO during seed maturation and germination for all genes present on the array. The time course included 12, 15, 18, and 20 days after flowering (DAF) from this study and after-ripened dry seeds (0) as well as seeds imbibed for 6, 26, 48, and 72 h (HAI) from the study by Bai et al, (2017)_. The average PO of the three biological replicates is plotted. The colours represent relative PO on a log₂ scale as indicated below the heat map. The 12 clusters are represented with Roman numerals. The number of genes in each cluster is indicated in parentheses. The grey bars that separate the developmental stages represent the three translational shifts [maturation translational shift (MTS), hydration translational shift (HTS), and germination translational shift (GTS)]. Heatmap colours indicate log₂ fold change from –3 (blue) to 3 (red) as indicated below the heatmap. All genes considered expressed are included in the heatmap. (B) Trend plot of PO across the whole time course for each of the clusters identified in (A). The maturation and germination stages are indicated with a blue and a green background, respectively.

Fig. 4.

SeedTransNet, a directional gene regulatory inference network based on changes in polysome occupancy (PO) during seed maturation and germination. The data for the network are combined from data presented in this study and the data from Bai et al. (2017). (A) SeedTransNet, with nodes representing genes and edges representing pairwise PO linkage. Colour indicates the modules detected from SeedTransNet; the major modules are colour coded. (B–D) Module topology of the top three modules. Nodes with high connectivity within each module are depicted as larger circles. Module 1 includes 334 genes with 1799 gene linkage inferences (GLIs) and an average module node degree of 10.78. Module 2 contains 364 genes and 684 GLIs with an average module node degree of 6.91. Module 3 consists of 140 genes representing 840 GLIs with an average module node degree of 4. (E) Transcript level and PO plotted across seed maturation (blue) and germination (green). The relative levels of the top 10 nodes from each of the top three modules are displayed. Blue lines represent PO and red lines indicate the transcript level in total RNA preparations.

Fig. 5.

SeedTransNet regulatory network hub genes affect seed characteristics. (A) Seed longevity phenotypes for plants with mutations in hub genes selected from Module 3. Five and seven days of artificial ageing were used to evaluate seed longevity in comparison with zero days of ageing. Error bars indicate average ±SD (n=4); asterisks indicate P-value <0.05 (Student’s t-test) as compared with Col-0 seeds. (B) Seed dormancy phenotypes of plants with mutations in hub genes selected from Module 3 represented by DSDS50 (the number of days of seed dry storage required to reach 50% germination). Error bars indicate average ±SD (n=4); asterisks indicate P-value <0.05 (Student’s t-test). (C) Close-up of a subset of the directional network in Module 3, centred around PM19L1, AT1G04560, and the target gene AT2G05580, Glycine-Rich Protein, which was confirmed experimentally (both red). Strengths of the predictions are indicated by edge thickness. (D) Ribosome occupancy (RO, ribosomal mRNA/total mRNA) of the downstream transcript targets (bold italic) of the predicted nodes from (C) is different in response to imbibition in wild-type and in mutant seeds (underlined) of the respective upstream nodes. Error bars indicate the average ±SD (n=3), P-value <0.05 (t-test).

Fig. 6.

Model summarizing the results of this study. The centre shows the seed translational network (SeedTransNet) that was built based on the polysome occupancy (PO) data obtained after polysome analysis at six stages from seed maturation (four time points) until seedling establishment (five time points). The different developmental stages are presented in the inner ring, and the polysome profiles on the outer ring. The red square indicates the seed maturation module, with a zoom in to PM19L1, a key regulator identified in the network that affects the translation of a glycine-rich transcript. Seed dormancy and longevity are determined during seed maturation; regulators of seed maturation therefore often contribute to dormancy and longevity phenotypes. This is also reflected in the phenotype of the pm19l1 mutant seeds (Fig. 5).