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.

Figure 1. Effects of light/dark transitions on At-RS31 alternative splicing

A) At-RS31 splicing event. *, PTC: premature termination codon. Triangles: primers for splicing evaluation (see also Figure S10a). B) Arabidopsis thaliana seedlings were incubated in light/dark for different times (Suppl. Text). C) After 48 hr of darkness, transferring the seedlings to light causes a rapid change in the SI of At-RS31 to “light values” (Suppl. Text). Light+Light, 48 hr + 3 hr light; Dark+Dark, 48 hr + 3 hr dark; Dark+Light, 48 hr dark + 3 hr light. D) Seedlings were grown for 2 weeks under short day conditions (~100 μmol/m²s). Samples were collected 2 hr before lights off (6 hr, zt) and 2 hr before lights on (22 hr, zt). E) Plants were grown in constant light, transferred to dark for 48 hr and then treated in light/dark for 6 hr with LEDs to provide specific wavelengths (Suppl. Text). F) hyh and hy5 mutants for integrators of photoreceptor-signaling pathways show the same At-RS31 alternative splicing regulation by light as WT plants (WS -Wassilewskija ecotype). G) Actinomycin D (ActD) causes the loss of the light/dark transition effect. ActD (+) was added 2 hr before light/dark treatments. DMSO, control (−). H) SI change induced by the light/dark transition is preserved in the NMD impaired mutants upf1-5 and upf3-1. I) Effect of U2AF⁶⁵ over-expression in A. thaliana protoplasts. In all experiments: white bars, light; black bars, darkness. Data represent means ± standard deviation (n≥3); significant p-values (Student’s t-test) are shown.

Figure 2. At-RS31 alternative splicing regulation is important for proper adjustment to light changes

A) qPCR analysis of At-RS31 mRNA1 levels relative to Actin. The graph shows the relative expression of mRNA1 in two different time points of light/dark treatment (2 and 4 hr). B) qRT-PCR analysis of At-RS31 mRNA1 isoform expression in the different genotypes and treatments. C) Top, At-RS31 SI in the different genotypes in response to light/dark (Suppl. Text); Bottom, representative gel images for the alternative splicing pattern of At-RS31 in the different genotypes in response to light/dark transitions. Actin, as control. L, light; D, dark. D) All lines were grown on MS agar plates with 1 % sucrose for 1 week in light/dark cyles (16/8 hr), 120 μmol/m²sec white light. Similar size sections of plates are shown. E) Seedlings of the different genotypes were grown under a 16/8 hr light/dark regime for 2 weeks and then transferred to dark for 3 days (D) or kept in photoperiod as controls (L). F) Seedlings for each genotype were grown on MS plates in constant light conditions (~50 μmol/m²sec). Similar size sections of plates are shown. Bar color code and statistics as in Figure 1.

Figure 3. The light signal is generated in the photosynthetic tissue and travels through the plant

A) Seedlings were grown in constant light, transferred to darkness for 48 hr and then treated with DCMU during a 6 hr light/dark further incubation. B-G) Light regulation of At-RS31 alternative splicing in green tissues and roots after a 2, 4 or 6 hr light/dark exposure of whole seedlings (B, D, F) or in the isolated parts exposed separately to light or dark (C, E, G). B, C) Schemes for post- and pre-exposure dissections (see Suppl. Text). D, F) At-RS31 alternative splicing assessment in green tissue (D) or in the roots (F) of light/dark treatments performed using intact seedlings (dissection performed after light/dark treatment). E, G) At-RS31 alternative splicing assessment in response to light/dark treatments using pre-dissected (E) green tissue or (G) roots (dissection performed before light/dark treatment). Bar color code and statistics as in Figure 1.

Figure 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 μM DBMIB to seedlings under medium (80 μmol/m² sec) and low (15 μmol/m² sec) light on At-RS31 alternative splicing. C-E) Addition of 30 μM 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 Figure 1.