Light regulates widespread plant alternative polyadenylation through the chloroplast

Abstract

Transcription of eukaryotic protein-coding genes generates immature mRNAs that are subjected to a series of processing events, including capping, splicing, cleavage, and polyadenylation (CPA), and chemical modifications of bases. Alternative polyadenylation (APA) greatly contributes to mRNA diversity in the cell. By determining the length of the 3' untranslated region, APA generates transcripts with different regulatory elements, such as miRNA and RBP binding sites, which can influence mRNA stability, turnover, and translation. In the model plant Arabidopsis thaliana, APA is involved in the control of seed dormancy and flowering. In view of the physiological importance of APA in plants, we decided to investigate the effects of light/dark conditions and compare the underlying mechanisms to those elucidated for alternative splicing (AS). We found that light controls APA in approximately 30% of Arabidopsis genes. Similar to AS, the effect of light on APA requires functional chloroplasts, is not affected in mutants of the phytochrome and cryptochrome photoreceptor pathways, and is observed in roots only when the communication with the photosynthetic tissues is not interrupted. Furthermore, mitochondrial and TOR kinase activities are necessary for the effect of light. However, unlike AS, coupling with transcriptional elongation does not seem to be involved since light-dependent APA regulation is neither abolished in mutants of the TFIIS transcript elongation factor nor universally affected by chromatin relaxation caused by histone deacetylase inhibition. Instead, regulation seems to correlate with changes in the abundance of constitutive CPA factors, also mediated by the chloroplast.

Keywords: Arabidopsis thaliana; alternative polyadenylation; light control in plants.

Fig. 1.

Light/dark conditions elicit widespread APA changes in Arabidopsis thaliana. (A) Protocol scheme of the light/dark regime used in this study. Total RNA from seedlings was subject to 3’READS for gene expression and APA analyses. (B) Distribution of identified PASs (Left) and PAS reads (Right) in different regions of the plant genome. (C) Different types of APA genes identified in this study. (D) Top, schematic of 3’UTR APA isoform; bottom, 3’UTR sizes for mRNAs of genes without 3’UTR APA (single 3’UTR) and mRNAs with the longest or shortest 3’UTRs of genes with 3’UTR APA. The median value for each group is indicated. (E) Diagram showing 3′UTR APA analysis. The two most abundant APA isoforms per gene were selected for comparison, which are named proximal PAS (pPAS) and distal PAS (dPAS) isoforms, respectively. The distance between the two PASs is considered alternative 3′UTR (aUTR). (F) Scatterplot showing genes with pPAS and dPAS isoform abundance differences between light- and dark-treated seedlings. Results represent analyses of 12,873 genes in three biological replicates. Genes with significantly (FDR < 0.05, DEXseq analysis) higher or lower abundance of pPAS isoforms in light vs. dark conditions are shown in blue and red, respectively.

Fig. 2.

The light effect on APA is sensed by the chloroplast (genetic and biochemical evidence). (A) 3’READS data for two Arabidopsis genes with opposite APA changes. Top: a representative event [HTA9 gene (AT1G52740), reads in red] with higher usage of dPAS in the light. Bottom: a representative event [RKH gene (AT5G15270), reads in blue] with higher usage of pPAS in the light. For each APA event, two pairs of primers were designed to validate the APA changes using RT-qPCR: amplicon dPAS (dark orange) only exists if the dPAS is used (long isoform); amplicon cod is common to all isoforms in the upstream coding region. Changes in APA are quantified as ratios of dPAS/cod amplicons relative mRNA expression levels for every gene. (B–D) Light is not sensed by photoreceptors. APA response to light/dark in different Arabidopsis phytochrome and cryptochrome mutant genotypes in a Landsberg erecta background (wt, Ler). Three selected genes are shown: HTA9, whose APA events increases its dPAS usage in the light (A), RKH, whose APA event diminishes its dPAS usage in the light (B) and FAD6, whose APA event is not affected by the light/dark conditions and serves as a negative control. (E–G) Effect of the photosynthetic electron transfer chain inhibitor DCMU on the light/dark effect on APA events of the HTA9 (E), RKH (F), and FAD6 (G) genes. Seedlings were grown in constant light, transferred to darkness for 48 h. and then treated with 20 µM DCMU during a 6-h. light/dark further incubation. RT-qPCR experiments were quantified with n ≥ 3, where n = ~25 to 30 Arabidopsis seedlings growing in one Petri dish. White and black bars represent light and dark treatments respectively. Changes considered significant show differences with a P value < 0.05 (two-tailed Student’s t test). ***P < 0.001; **P < 0.01; *P < 0.05; NS (not significant) = P > 0.05.

Fig. 3.

The light effect on APA is sensed by the photosynthetic tissues (anatomical evidence). (A and B) Schemes for dissections of Arabidopsis seedlings performed after (post-) or before (pre-) the light/dark treatment. (C–E) APA isoform analysis in green tissue (shoots) and in the roots of post- (Left) and pre-dissection (Right) light/dark treatments of the HTA9 (C), RKH (D), and FAD6 (E) genes. Bar colors and RT-qPCR conditions were as in Fig. 2.

Fig. 4.

Sugars, mitochondrial activity, and TOR kinase modulate the light effect on APA. HTA9 gene APA isoform analysis in green tissues (shoots) and roots of Arabidopsis seedlings dissected post- (A, C and D) and pre- (B) light/dark treatments. (A and B) Incubations were performed with 100 mM sucrose or sorbitol (negative osmotic control) as indicated. (C) Incubations were performed with 20 µM of the mitochondrial uncoupler dinitrophenol (DNP) or vehicle (ctrl.) as indicated. (D) Seedling were incubated with TOR kinase inhibitor AZD or vehicle (ctrl.) as indicated. Bar colors and RT-qPCR conditions were as in Fig. 2.

Fig. 5.

AS and APA respond differently to factors affecting transcript elongation. Effects of genetic disruption (tfiis mutant) of the transcription elongation factor TFIIS (A, B and C) and of treatment with the histone deacetylase inhibitor TSA (D, E and F) on AS of the Arabidopsis RS31 gene (A and D) and on APA of the HTA9 (B and E) and FAD6 (C and F) genes. Bar colors and RT-qPCR conditions were as in Fig. 2.

Fig. 6.

Light upregulates mRNA levels of cleavage/polyadenylation factors through the chloroplast. (A and B) RT-qPCR quantification of mRNA levels encoding subunits of CPSF (A) and CstF (B) in Arabidopsis shoots and roots obtained in a post-light/dark treatment excision experiment. (C and D) Effect of the photosynthetic electron transfer chain inhibitor DCMU on the light/dark effect on cleavage/polyadenylation factor mRNA levels in whole seedlings for CPSF (C) and CstF (D). Factor mRNA levels were relativized to mRNA levels of protein phosphatase 2A (PP2A). Bar colors and RT-qPCR conditions were as in Fig. 2.