Genome-wide network model capturing seed germination reveals coordinated regulation of plant cellular phase transitions

Abstract

Seed germination is a complex trait of key ecological and agronomic significance. Few genetic factors regulating germination have been identified, and the means by which their concerted action controls this developmental process remains largely unknown. Using publicly available gene expression data from Arabidopsis thaliana, we generated a condition-dependent network model of global transcriptional interactions (SeedNet) that shows evidence of evolutionary conservation in flowering plants. The topology of the SeedNet graph reflects the biological process, including two state-dependent sets of interactions associated with dormancy or germination. SeedNet highlights interactions between known regulators of this process and predicts the germination-associated function of uncharacterized hub nodes connected to them with 50% accuracy. An intermediate transition region between the dormancy and germination subdomains is enriched with genes involved in cellular phase transitions. The phase transition regulators SERRATE and EARLY FLOWERING IN SHORT DAYS from this region affect seed germination, indicating that conserved mechanisms control transitions in cell identity in plants. The SeedNet dormancy region is strongly associated with vegetative abiotic stress response genes. These data suggest that seed dormancy, an adaptive trait that arose evolutionarily late, evolved by coopting existing genetic pathways regulating cellular phase transition and abiotic stress. SeedNet is available as a community resource (http://vseed.nottingham.ac.uk) to aid dissection of this complex trait and gene function in diverse processes.

Fig. 1.

Properties and topologies of gene coexpression networks in Arabidopsis seeds. (A) Coexpression matrix showing gene interactions with a correlation coefficient above 0.6 (>0.75 in Fig. S2). Cluster 1 is highlighted by a red box and corresponds to an abundance of 50 randomly chosen SAM NG genes plotted within the matrix (red dots). Blue boxes indicate the location of clusters 2 and 3, which are associated with a high concentration of SAM G genes (blue dots). (B) Plot of the covariance breakdown for 100,000 randomly selected gene pairs. Black dots represent total covariance, blue dots represent covariance caused by preferential expression, and red dots represent covariance caused by other factors. (C) The SeedNet unweighted seed gene coexpression network. Regions 1, 2, and 3 are outlined in yellow and correspond to clusters 1, 2, and 3 in A. WGCNA network using all seed samples (D), only nongerminating seed samples (E), and only germinating seed samples (F). In all graphs, circles represent genes and lines represent significant transcriptional interactions between the genes. SAM NG genes are colored red and SAM G genes colored blue. Gray nodes are not classified by either gene list. All networks are displayed using Cytoscape organic layout.

Fig. 2.

Examination of SeedNet topology using known concepts of the regulation of seed germination. (A) Distribution of genes associated with dormancy (green) and germination (blue) (9, 16) (B) Distribution of after-ripening up-regulated (purple) and down-regulated (orange) genes (13). (C) Distribution of ABA up-regulated (tawny) and down-regulated (blue) genes (13). (D) Distribution of GA up-regulated (pink) and down-regulated (green) genes (11). (E) Distribution of genes up-regulated by drought (purple) and also on the SAM NG list (green) in Arabidopsis leaves (25). (F) Genes regulated by seed dormancy in wheat embryos. Dormancy up-regulated genes (pink) and germination-associated genes (green). (G) Distribution of genes involved in the developmental and hormonal regulation of seed germination within the 10 most significant modules identified by MCODE within SeedNet.

Fig. 3.

Transcriptional control of known regulatory genes, identification and function of regulators, and molecular validation of functional interactions. (A) Distribution of known dormancy and germination regulatory genes in SeedNet. Genes promoting dormancy are colored red and those promoting germination are colored blue. (B) Interactions between previously described and newly identified germination regulatory genes within the dormancy network located within the black box in A. Genes promoting dormancy are red, and those promoting germination are blue. Previously described regulators are labeled with black text and newly identified regulators are in dark blue. Known interactions are indicated by green edges and newly identified interactions (from G and H) by pink edges. Increased node size corresponds to higher degree and edge width to interaction strength. (C) Dose response of newly characterized mutant seeds to exogenous ABA. (D) Same as C using the GA synthesis inhibiting compound paclobutrazol (PAC). (E) Germination of mutant seeds at 10 °C. (F) Germination of agl67 mutant seeds 2 wk after harvest assayed directly (not chilled) or following 48 h of cold treatment at 4 °C to remove dormancy (chilled). (G) Increased stability of ABI3 and ABI5 proteins in the scl14-1 mutant in the presence of 0.5 μM ABA. (H) Increased stability of ABI5 protein in asg2-1 in the presence of 0.5 μM ABA. (I) Interactions predicted by AraNet between the genes in the graph in B. Interactions common to both networks are represented by red colored edges. Data in C–F are means from four independent experiments; error bars show SD.

Fig. 4.

The developmental phase transition between nongermination and germination is captured by SeedNet. (A) Heat map shows the relative transcript abundance of key regulators of dormancy and germination during the time course of seed germination. Hours after imbibition are indicated above the map. (B) Heat map of representative genes within different regions of the network labeled with black circles in C and a lowercase letter to the right of the heat map. Scale below heat maps in A and B indicates log₂-transformed transcript abundance relative to median expression on a gene-by-gene basis. (C) Network with nodes corresponding to region 2-specific modules 52 (red) and 58 (blue) highlighted. (D) Seed dormancy phenotype of efs. Different seeds harvested from efs mutant plants are shown, and the percentage of seeds with an altered phenotype are indicated below each image, relative to WT (Ler). (Scale bar: 100 μm.) (E) Response of serrate (se) mutants seeds to increasing concentrations of ABA relative to WT (Col). Data in E are means from four independent experiments; error bars show SD.