Extensive translational regulation during seed germination revealed by polysomal profiling

Abstract

This work investigates the extent of translational regulation during seed germination. The polysome occupancy of each gene is determined by genome-wide profiling of total mRNA and polysome-associated mRNA. This reveals extensive translational regulation during Arabidopsis thaliana seed germination. The polysome occupancy of thousands of individual mRNAs changes to a large extent during the germination process. Intriguingly, these changes are restricted to two temporal phases (shifts) during germination, seed hydration and germination. Sequence features, such as upstream open reading frame number, transcript length, mRNA stability, secondary structures, and the presence and location of specific motifs correlated with this translational regulation. These features differed significantly between the two shifts, indicating that independent mechanisms regulate translation during seed germination. This study reveals substantial translational dynamics during seed germination and identifies development-dependent sequence features and cis elements that correlate with the translation control, uncovering a novel and important layer of gene regulation during seed germination.

Keywords: Arabidopsis; RNA structure; germination; imbibition; polysomal profiling; ribosome; seedling establishment; translatomics.

Figure 1

Polysome profiling during seed germination. (a) Frequency of testa rupture (TR), radicle protrusion (RP) and seedling greening (SG) during the Arabidopsis Col‐0 seed to seedling transition. Time points during seed germination include dry seeds, 6, 26 (TR initiation), 48 (80% RP) and 72 h after the start of imbibition (HAI) (80% GS). Data are presented as mean ± SD of three independent replicates. (b) Absorbance profiles of sucrose density gradient fractionated ribosomes for the five time points during the seed to seedling transition. Data are presented as mean ± SD of three independent replicates. SD is indicated by the width of the line. Gradient loading according to equal dry weight. (c) Representative absorbance profiles of sucrose density gradient fractionated ribosomes for the five time points during the seed to seedling transition; gradient loading according to identical RNA loading. (d) Ribosome abundance for the five time points during the seed to seedling transition. Data are presented as mean ± SD of three independent replicates and the letters above each bar indicate the significance (P < 0.05). (e) Relative abundance of different ribosomes (Cyt, cytosolic; Pla, plastidic; Mit, mitochondrial).

Figure 2

Arabidopsis seed germination is characterized by two translational shifts. (a) Principal component (PC) analysis of polysome occupancy (PO) changes (polysomal mRNA levels/total mRNA levels) during seed germination. The first two components (PC1 and PC2) explain 30 and 13% of the total variation, respectively. (b) The number of mRNAs with changed PO at the two translational shifts, at 6 h after the start of imbibition (HAI) the hydration translational shift (HTS) and 48 HAI, the germination translational shift (GTS). (c) Dynamics of genes underlying the two translational shifts. The levels of total (x‐axis) and polysomal (y‐axis) mRNAs following seed imbibition (dry seeds, 6, 26, 48 and 72 HAI) are plotted (black). Genes identified with increased (red dots) or decreased (green dots) polysome occupancy are indicated for both the HTS (upper panels) and the GTS (lower panels).

Figure 3

Total RNA and polysomal RNA profile of translational regulated genes during the seed to seedling transition in Arabidopsis. Translational regulated genes are divided into subgroups based on their total RNA and polysomal RNA changes. In total, 22 subgroups under nine categories are identified with their corresponding expression profiles following seed germination and genes numbers under each subgroup in parentheses. T+ represents that the transcription is increased while the translation is unchanged under a specific shift. P+ indicates translation is increased while the total mRNA abundance is unchanged. T+/P+ indicates both transcription and translation are enhanced and an additive effect means that the change of gene expression in either transcription or translation is not significant while the additive effect of the two contributes to the significant change of polysome occupancy. The opposite effect is represented by T−, P− and T−/P− in the corresponding subgroups. HTS, hydration translational shift; GTS, germination translational shift; HAI, hours after the start of imbibition.

Figure 4

mRNAs of the two shifts during the seed to seedling transition in Arabidopsis are characterized by distinct sequence features. (a) Length of coding DNA sequence (CDS), (b) GC content of 5′UTR, (c) GC3 content and (d) effective codon number (N _c) of the CDS are shown. Colour scheme: black, microarray background (Bkg); red or green, translationally up‐ or downregulated genes at the hydration translational shift (HTS) and germination translational shift (GTS), respectively. Error bars represent SE (*, P < E‐10; **, P < E‐20; ***, P < E‐50). P‐values are calculated by a Wilcoxon nonparametric test.

Figure 5

Secondary structures and motif features correlate with polysome association. The average structure score is plotted over the 5′UTR, coding DNA sequence (CDS) and 3′UTR of the (a) hydration translational shift (HTS) and (b) germination translational shift (GTS) affected transcripts. Background: all transcripts on the array (black line), translational up (red line) or translational down (green line). P‐values are calculated by a Student's test according to Li et al. (2012). The bar plots represent the mean structure scores per transcript region (5′UTR, CDS and 3′UTR, respectively). Error bars represent SE (*, P < E‐10; ***, P < E‐50; ****, P < E‐100 ). Significantly enriched motifs are detected across (c) the whole transcript length (cDNA) and (d) 5′UTR. P‐values are calculated by a Fisher's t‐test. Note: several sequence motifs overlapping with motif 2b were identified. Only the most significant is depicted.

Figure 6

Summary of the sequence features in mRNAs identified as translationally regulated during germination. The upper panel shows a schematic presentation of the transition from a dry seed to a seedling at 72 h after the start of imbibition (HAI). The two phases where large sets of mRNAs are under translational control, the hydration translational shift (HTS) and germination translational shift (GTS), are indicated. The identified features that correlate with these translational shifts are listed. CDS, coding DNA sequence; uORF, upstream open reading frame; UC, pyrimidine; GA, purine.