A Chloroplast Retrograde Signal Regulates Nuclear Alternative Splicing

Abstract

Light is a source of energy and also a regulator of plant physiological adaptations. We show here that light/dark conditions affect alternative splicing of a subset of Arabidopsis genes preferentially encoding proteins involved in RNA processing. The effect requires functional chloroplasts and is also observed in roots when the communication with the photosynthetic tissues is not interrupted, suggesting that a signaling molecule travels through the plant. Using photosynthetic electron transfer inhibitors with different mechanisms of action we deduce that the reduced pool of plastoquinones initiates a chloroplast retrograde signaling that regulates nuclear alternative splicing and is necessary for proper plant responses to varying light conditions.Light regulates approximately 20% of the transcriptome in Arabidopsis thaliana and rice (1, 2). Alternative splicing has been shown to modulate gene expression during plant development and in response to environmental cues (3). We observed that the alternative splicing of At-RS31 ( Figure 1A), encoding a Ser-Arg-rich splicing factor (4), changed in different light regimes, which led us to investigate how light regulates alternative splicing in plants. Figure 1B). This effect was rapidly reversed when seedlings were placed back in light, with total recovery of the original SI in about 3 hr ( Figure 1C), indicating that the kinetics of the splicing response is slower from light to dark than from dark to light.The light effect is gene-specific ( Figure S1) and is also observed in diurnal cycles under short day conditions ( Figures 1D and S2). Furthermore, three circadian clock mutants behaved like the wild type (WT) in the response of At-RS31 alternative splicing to light/dark ( Figure S3). Changes in At-RS31 splicing are proportional to light intensity both under constant light or in short day grown seedlings ( Figure S4).Both red (660 nm) and blue (470 nm) lights produced similar results as white light ( Figure 1E). Moreover, At-RS31 splicing is not affected in phytochrome and cryptochrome signaling mutants (5, 6) behave as WT seedlings, ruling out photosensory pathways in this light regulation (Figures 1F, S5 and S6).Light-triggered changes in At-RS31 mRNA patterns are not due to differential mRNA degradation. First, the light effect is not observed in the presence of the transcription inhibitor actinomycin D ( Figure 1G). Second, the effects are still observed in upf mutants, defective in the nonsense-mediated mRNA decay (NMD) pathway (7) mRNA1 is the only isoform encoding a full-length At-RS31 protein (9). mRNA3 and mRNA2 are almost fully retained in the nucleus ( Figure S8). mRNA1 levels decrease considerably in dark without significant changes in the total amount of At-RS31 transcripts (Figures 2A and S9) which suggests that alternative splicing is instrumental in the control of mRNA1 cellular levels and, consequently, At-RS31 protein abundance. To assess how interference with At-RS31 alternative splicing regulation could affect Arabidopsis phenotype we ...

Phytochrome controls alternative splicing to mediate light responses in Arabidopsis

“…analyzed the overlapping target genes that are regulated by both phytochrome and chloroplast retrograde signaling pathways and found that there is basically no correlation between these two pathways (Table S5), although they are not mutually exclusive and may coexist and act coordinately in certain conditions, sharing common target genes, such as RS31 (35). GO and other statistical analyses suggested that phytochromemediated control of not only TX, but also AS, have significant roles in light signaling (Fig.…”

“…Therefore, phytochrome-mediated AS regulation that we demonstrated here seems to be widely conserved in plants. It has been reported very recently that light affects AS of a subset of Arabidopsis genes through a chloroplast retrograde signal, but not through photoreceptor signaling (35). We thus Four-dayold etiolated seedlings were exposed to a 2-min pR (30 μmol·m −2 ·s −1 ), 2-min pFR (60 μmol·m −2 ·s −1 ), pR followed by subsequent pFR (pR/pFR), or were kept in darkness (D).…”

Retrograde Signals Navigate the Path to Chloroplast Development

“…Proper splicing of these 319 factors was proposed to play a role in the adaptation of the photosynthetic machinery to changes 320 in the light conditions (Petrillo et al, 2014). However, more experimental work is required to 321 identify the components that regulate splicing in response to retrograde signals, the genes 322 affected, and how those changes affect plant development and photosynthetic activity.…”

“…Light has been shown to induce changes in splicing activity (Petrillo et al, 2014;Shikata et al, 317 2014;Hernando et al, 2017) and the splicing pattern of specific splicing factors involved in 318 dark-to-light shifts is regulated in response to the redox status of PET. Proper splicing of these 319 factors was proposed to play a role in the adaptation of the photosynthetic machinery to changes 320 in the light conditions (Petrillo et al, 2014).…”

Chloroplasts extend stromules independently and in response to internal redox signals

“…In recent years, forward genetic screens have led to the discoveries that chloroplasts are critical regulators of leaf shape, cell-cell signaling through plasmodesmata, pathogen defense, and even alternative splicing in the nucleus (1)(2)(3)(4)(5)(6)(7)(8); however, in almost all of these pathways, the signaling route between the chloroplast and the nucleus is unknown. This is a pressing question for plant biology and cell biology in general: how do organelles communicate with the nucleus to coordinate genetic programs and cellular function?…”