associated with the energy state of the cells (fig.
S16a). In addition, we ruled out the participation of
sugar-sensing pathways (fig. S16, b and c) reported
to control nuclear gene expression (12, 13).
Analysis of light-responsive splicing events revealed an enrichment in those of RNA-binding
and processing protein coding genes (fig. S17 and
table S3). The SR protein genes At-SR30 and
At-U2AF65 showed the biggest changes (fig. S18
and table S2), and DCMU treatment also confirmed
a role for chloroplast involvement in their responses
Experiments shown in figs. S20 and S21 revealed that plastid gene expression, the tetrapyrrole
pathway, and reactive oxygen species (14) are not
involved in At-RS31 alternative splicing regulation. Because methyl viologen takes electrons from
PSI (15) with no effect on At-RS31 alternative
splicing (fig. S21a), we inferred that the signal
must be generated between PSII and PSI. To prove
this, we used DBMIB (2,5-dibromo-3-methyl-6-
isopropyl-p-benzoquinone). Both DCMU and
DBMIB inhibit the overall electron transport chain,
but whereas DCMU increases the oxidized PQ
pool by blocking the electron transfer from the
PSII to PQ (10), DBMIB keeps the PQ pool reduced by preventing the electron transfer to cytochrome b6/f (15) (Fig. 4A). Addition of DBMIB
potentiates the decrease in At-RS31 SI when
seedlings are exposed to low light in comparison
to the lack of effect under high light (Fig. 4B).
It was shown that when externally added in the
dark, DBMIB can act as a reduced quinone an-
alog (16, 17). Consistently, DBMIB decreases
At-RS31 SI in the dark, mimicking the effects
of light (Fig. 4C). Similar results were obtained
for At-U2AF65 and At-SR30 (Figs. 4, D and E).
Our results reveal a retrograde pathway linking the photosynthetic redox state to the regulation of nuclear alternative splicing, mediated by
the PQ pool, together with a signaling molecule, of
yet unknown nature, that is able to travel through
the plant to affect alternative splicing (Fig. 4F).
References and Notes
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Acknowledgments: We thank J. Casal, J. Estévez, N. Iusem,
J. Palermo, G. Cabrera, G. Siless, N. Carrillo, F. Rolland,
J. Chory, J.-S. Jeon, R. Hausler, J. Sheen, A. Köhler, A. Trebst,
and S. Smeekens for materials and advice. This work was
supported by the Agencia Nacional de Promoción de Ciencia y
Tecnología of Argentina, the University of Buenos Aires, the
King Abdulaziz University, the European Network on
Alternative Splicing, the Austrian Science Fund FWF (P26333
to M.K.; DK W1207, SFBF43-P10, ERA-NET I254 to A.B.),
the U.K. Biotechnology and Biological Sciences Research
Council, and the Scottish Government Rural and Environment
Science and Analytical Services division. M.G.H. is a
fellow and A.R.K. is a career investigator from the Consejo
Nacional de Investigaciones Científicas y Técnicas of
Argentina. E.P. is an EMBO postdoctoral fellow. A.R.K. is a
senior international research scholar of the Howard Hughes
Medical Institute (for detailed acknowledgments, see the
Materials and Methods
Figs. S1 to S22
Tables S1 to S4
2 January 2014; accepted 28 March 2014
Published online 10 April 2014;
C D E F ROOT SHOOT
Fig. 4. The plastoquinone redox state mediates alternative splicing
regulation by light. (A) Diagram showing the action of DBMIB and DCMU
in the photosynthetic electron transport chain. (B) Effects of the addition
of 30 mM DBMIB to seedlings under medium (80 mmol m–2 s–1) and low
(15 mmol m–2 s–1) light on At-RS31 alternative splicing. (C to E) Addition
of 30 mM DBMIB reduces the effects of light/dark transitions on At-RS31
(C), At-U2AF65 (D), and At-SR30 (E) alternative splicing patterns. (F) Model
for the light regulation of alternative splicing. Light-induced reduction of
plastoquinone to plastoquinol (PQH2) generates a signal that modulates
alternative splicing in the nucleus. This signal, or a derived one, travels to
the roots and provokes similar effects. Bar color code and statistics as in