Processing bodies control the selective translation for optimal development of Arabidopsis young seedlings

Abstract

Germinated plant seeds buried in soil undergo skotomorphogenic development before emergence to reach the light environment. Young seedlings transitioning from dark to light undergo photomorphogenic development. During photomorphogenesis, light alters the transcriptome and enhances the translation of thousands of mRNAs during the dark-to-light transition in Arabidopsis young seedlings. About 1,500 of these mRNAs have comparable abundance before and after light treatment, which implies widespread translational repression in dark-grown seedlings. Processing bodies (p-bodies), the cytoplasmic granules found in diverse organisms, can balance the storage, degradation, and translation of mRNAs. However, the function of p-bodies in translation control remains largely unknown in plants. Here we found that an Arabidopsis mutant defective in p-body formation (Decapping 5; dcp5-1) showed reduced fitness under both dark and light conditions. Comparative transcriptome and translatome analyses of wild-type and dcp5-1 seedlings revealed that p-bodies can attenuate the premature translation of specific mRNAs in the dark, including those encoding enzymes for protochlorophyllide synthesis and PIN-LIKES3 for auxin-dependent apical hook opening. When the seedlings protrude from soil, light perception by photoreceptors triggers a reduced accumulation of p-bodies to release the translationally stalled mRNAs for active translation of mRNAs encoding proteins needed for photomorphogenesis. Our data support a key role for p-bodies in translation repression, an essential mechanism for proper skotomorphogenesis and timely photomorphogenesis in seedlings.

Keywords: light; p-bodies; photomorphogenesis; skotomorphogenesis; translation.

Fig. 1.

dcp5-1 mutant is hypersensitive to light. (A) Representative images of 4-d-old WT, dcp5-1, and dcp5-1 DCP5 complementation line grown under the dark or 7 μE white light. (B) Fluence response curves of 4-d-old WT, dcp5-1, and dcp5-1 DCP5 complementation line under dark, 4 μE, 7 μE, and 40 μE continuous white light (Wc). (C) Fluence response curves of normalized hypocotyl length to that of corresponding genotype grown under the dark condition. Asterisks in B and C indicate shorter hypocotyl length for dcp5-1 than the WT (Student’s t test; *P < 0.001). Data are mean ± SD from one representative experiment (n ≥ 30). Similar results were observed in three independent experiments.

Fig. 2.

Light reduces p-body accumulation. (A and B) Representative images of p-bodies observed in cotyledons of WT expressing DCP2-YFP. Enlarged images were shown in Insets at bottom left corners. DCP2-YFP foci number and relative foci area (normalized to the mean of Dark samples) were calculated as described in SI Appendix, SI Materials and Methods. Box and whisker plots from one representative experiment are shown [n = 7 (Dark); n = 6 (L4h)]. The top, middle, and bottom of the box represent the 25th, 50th and 75th percentiles, respectively. The whiskers are minimum and maximum. Similar results were observed in three independent experiments. Asterisk indicates significantly different number and size for p-bodies in L4h vs. the Dark samples (Student’s t test; *P < 0.005). (C, Left) shows protein level of DCP2-YFP in 4-d-old WT seedlings before and after light treatment for 1 or 4 h. (C, Right ) shows Coomassie blue-stained blot as a protein loading control. (D and E) Representative photographs and plots of DCP2-YFP in hy2-106 under Dark and L4h (n = 6). (F) DCP2-YFP in etiolated cop1-6 mutant and WT. (Scale bar, 10 μm.)

Fig. 3.

P-bodies repress massive translation in etiolated seedlings. (A) An illustration of the experimental design. Steady-state mRNAs (mRNA_SS) and polysome-bound (mRNA_PL) were isolated in parallel and hybridized to Affymetrix ATH1 GeneChips for transcriptomic profiling analyses. (B) PL% in WT and dcp5-1 under Dark and L4h conditions. Data are mean ± SEM from four biological replicates. Spike-in RNA (DAP) was used for data normalization. Asterisk indicates translation efficiency of conditions statistically different from that of 4-d-old etiolated WT seedlings (Student’s t test; *P < 0.05). (C) Classification of DCP5-regulated genes at mRNA_SS or mRNA_PL level in dcp5-1 normalized to the WT. K-means clustering was used to classify the 2,391 genes regulated by DCP5. Extreme yellow and blue colors indicate fourfold up-regulation and down-regulation, respectively.

Fig. 4.

DCP5 regulates the translation of genes encoding the chlorophyll biosynthetic enzymes. (A) A diagram of chlorophyll biosynthesis pathway. Genes with translation regulated by DCP5 are in orange. (B) Expression of mRNA_SS and mRNA_PL in dcp5-1 or WT plants under Dark or L4h. Data are mean ± SEM from three biological replicates. Asterisk indicates that the level of a given gene significantly differs in dcp5-1 vs. the WT (Student’s t test; *P < 0.05). (C) Pchlide level in etiolated WT and dcp5-1 seedlings. y axis marks relative fluorescence with arbitrary units (a.u.). (D) The representative photograph showing photobleaching phenotype was only observed in dcp5-1 seedlings.

Fig. 5.

DCP5 is required for skotomorphogenic development. (A) Representative photograph and box-and-whisker plot of apical hook angle of 4-d-old etiolated WT and dcp5-1 seedlings (n = 38–44). Asterisk indicates that the hook angle is larger for dcp5-1 than the WT (Student’s t test; *P < 0.001). (B) Representative photograph of WT and dcp5-1 after germination under 1 mm sand. (C) Protruding rate of WT and dcp5-1 seedlings grown with or without coverage of 1 mm sand. Data are mean ± SEM from three biological replicates (n = 98–126). Asterisk represents a much-reduced protrusion rate for dcp5-1 than the WT (Student’s t test; *P < 0.001). (D) Expression of PILS3 mRNA_SS and mRNA_PL in dcp5-1 or WT under Dark or L4h. Data are mean ± SEM from three biological replicates. Asterisk indicates that the level of PILS3 in dcp5-1 is statistically different from that in WT (Student’s t test; *P < 0.05). (E) A working model for translational regulation by p-bodies during de-etiolation. Image courtesy of Hsuan Pai (Academia Sinica, Taipei, Taiwan).