complex than shifting FEF signals between different locations in the visual field.
IFJ may include areas that function as general
executive modules (22, 23). Also, IFJ is close to
areas Ba45 and Ba46, homologs of which have
been described in nonhuman primate recordings
to encode information about object-categories in
delayed match-to-sample tasks (23, 24). Indeed,
the “attentional template” that specifies the relevant location or object in spatial or feature attention is hardly distinguishable from working
memory for these qualities (9), which is known to
involve prefrontal cortex (24). Coupled interactions between prefrontal areas and visual areas
(25–31) could underlie many cognitive phenomena in vision, with shared neural mechanisms
but variations in the site of origin and the site of
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Acknowledgments: We thank J. Liang, D. Pantazis,
M. Hämäläinen, D. Dilks, D. Osher, Y. Zhang, C. Triantafyllou,
S. Shannon, S. Arnold, C. Jennings. Supported by NIH
(P30EY2621) and NSF (CCF-1231216), both to R.D.
Materials and Methods
Figs. S1 to S13
8 October 2013; accepted 1 April 2014
Published online 10 April 2014;
A Chloroplast Retrograde Signal
Regulates Nuclear Alternative Splicing
Ezequiel Petrillo,1 Micaela A. Godoy Herz,1 Armin Fuchs,2 Dominik Reifer,2
John Fuller,3 Marcelo J. Yanovsky,4 Craig Simpson,3 John W. S. Brown,3,5
Andrea Barta,2 Maria Kalyna,2† Alberto R. Kornblihtt1‡
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 about 20% of the transcrip- tome in Arabidopsis thaliana and rice (1, 2). Alternative splicing has been shown
to modulate gene expression during plant devel-
opment and in response to environmental cues
(3). We observed that the alternative splicing
of At-RS31 (Fig. 1A), encoding a Ser-Arg–rich
splicing factor (4), changed in different light re-
gimes, which led us to investigate how light reg-
ulates alternative splicing in plants.
Seedlings were grown for a week in constant
white light to minimize interference from the circadian clock and then transferred to light or dark
conditions for different times (see the supplementary materials). We observed a two- and fourfold increase in the splicing index (SI)—defined
as the abundance of the longest splicing isoform
relative to the levels of all possible isoforms—of
At-RS31 [mRNA3/(mRNA1 + mRNA2 + mRNA3)]
after 24 and 48 hours in the dark, respectively
(Fig. 1B). This effect was rapidly reversed when
seedlings were placed back in light, with total
recovery of the original SI in about 3 hours (Fig.
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 (fig. S1) and
is also observed in diurnal cycles under short-day
conditions (Fig. 1D and fig. S2). Furthermore,
three circadian clock mutants behaved like the
wild type (WT) in the response of At-RS31 alternative splicing to light/dark (fig. S3). Changes
in At-RS31 splicing are proportional to light intensity both under constant light and in short-day–
grown seedlings (fig. S4).
Both red (660 nm) and blue (470 nm) lights
produced similar results as white light (Fig. 1E).
Moreover, At-RS31 alternative splicing responses
to light/dark are not affected in phytochrome and
cryptochrome signaling mutants (5, 6), ruling out
photosensory pathways in this light regulation
(Fig. 1F and figs. 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
(Fig. 1G). Second, the effects are still observed in
upf mutants, defective in the nonsense-mediated
mRNA decay (NMD) pathway (7) (Fig. 1H and
fig. S7). Third, overexpression of the constitutive
splicing factor U2AF65 (8) in Arabidopsis protoplasts mimics the effects of light on At-RS31 alternative splicing (Fig. 1I).
mRNA1 is the only isoform encoding a full-length At-RS31 protein (9). mRNA3 and mRNA2
are almost fully retained in the nucleus (fig. S8).
mRNA1 levels decrease considerably in dark without significant changes in the total amount of
At-RS31 transcripts (Fig. 2A and fig. S9), which
suggests that alternative splicing is instrumental
1Laboratorio de Fisiología y Biología Molecular, Departamento
de Fisiología, Biología Molecular y Celular, IFIBYNE-CONICET,
Facultad de Ciencias Exactas y Naturales, Universidad de
Buenos Aires, Ciudad Universitaria, Pabellón 2, C1428EHA
Buenos Aires, Argentina. 2Max F. Perutz Laboratories, Medical
University of Vienna, A-1030 Vienna, Austria. 3Cell and Molecular Sciences, The James Hutton Institute, Invergowrie, Dundee,
Scotland. 4Fundación Instituto Leloir, IIBBA-CONICET, C1405BWE
Buenos Aires, Argentina. 5Division of Plant Sciences, University of
Dundee at The James Hutton Institute, Invergowrie, Dundee,
*Present address: Max F. Perutz Laboratories, Medical University
of Vienna, A-1030 Vienna, Austria.
†Present address: Department of Applied Genetics and Cell
Biology, BOKU, University of Natural Resources and Life
Sciences, Muthgasse 18, A-1190 Vienna, Austria.
‡Corresponding author. E-mail: firstname.lastname@example.org