Photosynthetic complexes in chloroplasts are built from subunits encoded by two genomes: chloroplast-encoded subunits are translated by bacterial-type chlororibosomes, while nuclear-encoded subunits are translated in the cytosol and imported. Ribosome profiling across acute light/dark transitions (Wakigawa et al., 2024) showed that the two systems cope with light changes by different mechanisms: chloroplasts adjust translation, whereas the cytosol adjusts mRNA abundance, and that reduced plastoquinone (PQH2) triggers chloroplast translational activation while a distinct, unidentified retrograde signal controls cytosolic mRNA abundance. The open problem is the molecular identity of the machinery on both sides: (i) the retrograde signaling molecule(s) - among candidates reactive oxygen species, methylerythritol cyclodiphosphate, carotenoid derivatives, heme, and 3'-phosphoadenosine 5'-phosphate (PAP), or others - through which the chloroplast controls light-responsive cytosolic mRNA abundance, including whether PAP-mediated inhibition of the 5'-to-3' exonuclease XRN2 executes this regulation; (ii) whether the rapid loss of these cytosolic mRNAs in the dark reflects active mRNA decay, transcriptional tuning, or both; and (iii) the plastid machinery that detects PQH2 and the downstream effectors that activate translation on chloroplast mRNAs encoding complex subunits.

Background

Chloroplasts descended from an endosymbiotic cyanobacterium, and their photosynthetic complexes - photosystem II (PSII), cytochrome b6f, photosystem I (PSI), the FoF1 ATP synthase, and the NADH dehydrogenase-like (NDH) complex - are assembled from subunits encoded in two genomes. A few dozen core subunits are encoded in the chloroplast genome and translated by bacterial-type ribosomes (chlororibosomes) in the organelle, while the majority are encoded in the nucleus, translated in the cytosol, and imported. Because the subunits of each complex must accumulate stoichiometrically, plant cells must continuously coordinate the two gene-expression systems, particularly during light/dark transitions when demand for photosynthetic proteins changes abruptly.

Ribosome profiling - deep sequencing of ribosome-protected mRNA fragments, which yields a transcriptome-wide readout of translation - can simultaneously quantify translation in the cytosol and in chloroplasts because the two ribosome populations protect fragments of diagnostic lengths. Applying this approach to Arabidopsis, the grass Brachypodium, the green alga Chlamydomonas, the glaucophyte alga Cyanophora (which retains a primitive chloroplast), and the cyanobacterium Synechocystis, Wakigawa et al. (2024) found that land plants and green algae respond to acute light/dark shifts with a division of labor: in chloroplasts, protein output follows translational regulation (the mRNA pool changes little), whereas in the cytosol it follows changes in mRNA abundance. Cyanobacteria, the ancestors of chloroplasts, regulate mRNA abundance only, implying that chloroplast translational control arose with endosymbiosis.

Two pharmacological results pin down the upstream photosynthetic signal. 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), which blocks electron flow from PSII to plastoquinone (PQ) and thus prevents formation of reduced plastoquinone (plastoquinol, PQH2), attenuates light-induced translational activation in chloroplasts and the induction of cytosolic mRNAs encoding complex subunits. Conversely, 2,5-dibromo-3-methyl-6-isopropylbenzoquinone (DBMIB), which blocks PQH2 oxidation by cytochrome b6f and thereby keeps the PQ pool reduced, mimics light for chloroplast translation even in the dark - but does not induce the cytosolic mRNAs. The picture is that PQH2 itself activates chloroplast translation, whereas cytosolic mRNA abundance is governed by a separate retrograde signal - a molecule produced by the chloroplast that alters gene expression in the cytosol/nucleus.

A further pharmacological result constrains the direction of coordination: cycloheximide, which blocks cytosolic translation, also blocked the light-induced translational activation of chloroplast mRNAs in Arabidopsis and Chlamydomonas, whereas chloramphenicol, which blocks chloroplast translation, did not prevent the light-induced accumulation of cytosolic mRNAs encoding complex subunits. The authors concluded that coordination between the two gene-expression systems is unidirectional (cytoplasm to chloroplast), and suggested that the cytosolic translation requirement could be explained by de novo synthesis of transcriptional factors at the beginning of light exposure - a mechanism that remains open (Wakigawa et al., 2024).

Retrograde signaling is a long-studied layer of plant cell regulation. Known or proposed chloroplast-derived signals include reactive oxygen species (ROS), the isoprenoid precursor methylerythritol cyclodiphosphate (MEcPP), carotenoid-derived cleavage products, heme, and 3'-phosphoadenosine 5'-phosphate (PAP). PAP accumulates when its phosphatase SAL1 is inactivated, and inhibits the 5'-to-3' exoribonucleases XRN2 and XRN3 (and the cytosolic XRN4), a pathway documented in drought and high-light signaling and in the chloroplast control of nuclear microRNA biogenesis (Estavillo et al., 2011; Fang et al., 2019). Chloroplast-dependent, photoreceptor-independent regulation of nuclear RNA metabolism is well established - light controls alternative splicing and, more recently, alternative polyadenylation through functional chloroplasts (Petrillo et al., 2014; Kubaczka et al., 2024) - but the molecular identity of the signal behind these responses, and behind the light/dark control of cytosolic mRNA abundance, is not established (Zhao et al., 2020). Because the cytosolic mRNAs encoding chloroplast complex subunits fall within minutes of a light-to-dark shift, mRNA decay is the most likely effector mechanism, though a contribution from transcriptional tuning (reduced synthesis in the dark) has not been excluded; whether PAP-XRN2 participates is untested.

On the plastid side, light-dependent chloroplast translation is thought to be regulated mainly at initiation. The best-studied case is the psbA mRNA, encoding D1, the PSII reaction-center subunit that is continuously photodamaged and replaced: classic work showed light regulates D1 synthesis, including elongation control by the light-generated proton gradient (Mühlbauer and Eichacker, 1998), and 2025 studies dissected a psbA-specific initiation circuit in which the translational regulator HCF173 toggles between activator and repressor and the assembly factors RBD1, HCF136, and HCF244 link D1 photodamage and assembly state to translation rate (Williams-Carrier et al., 2025; Rojas et al., 2025), a scheme conserved in the liverwort Marchantia (Chotewutmontri et al., 2025). However, this circuit is driven by D1 photodamage rather than by PQH2 sensing, and Marchantia ribosome profiling also reveals a plastome-wide light activation of translation elongation whose mechanism is unexplained. Redox sensing of the PQ pool is meanwhile well established for other outputs - cytochrome b6f sensing controls the STN7 kinase in state transitions (light-state tuning of the antennae), and the chloroplast sensor kinase CSK couples plastoquinone redox state to plastid transcription (Ibrahim et al., 2020) - but no sensor connecting PQH2 to translational activation of the broader set of chloroplast mRNAs encoding ATP synthase, cytochrome b6f, PSI, and NDH subunits has been identified.

Two auxiliary open sub-questions complete the source's agenda. First, chlororibosomes pause at defined sites on psbA mRNA (notably after the fourth transmembrane segment of nascent D1), a phenomenon with interspecies variation whose functional significance - for complex assembly or protein quality control - is unresolved (Gawroński et al., 2018); disome profiling, the sequencing of collided ribosome pairs, has been proposed as the way to address it. Second, in cyanobacteria, longer dark exposures may repress translation through the stringent response mediated by (p)ppGpp (guanosine tetra- and pentaphosphate alarmone), which is required for dark survival in Synechococcus (Hood et al., 2016), but this was not tested for the short dark periods used in the source study.

Problem Statement

Identify the molecular machinery that synchronizes chloroplastic and cytosolic protein synthesis during light/dark transitions, as established by Wakigawa et al. (2024):

  1. The retrograde signaling molecule(s) - among the candidates reactive oxygen species, methylerythritol cyclodiphosphate (MEcPP), carotenoid derivatives, heme, and 3'-phosphoadenosine 5'-phosphate (PAP), or a molecule outside this list - by which the chloroplast controls the abundance of cytosolic mRNAs encoding chloroplastic complex subunits in response to light. This includes testing whether PAP-mediated inhibition of the 5'-to-3' RNA exonuclease XRN2 executes this regulation.

  2. The mechanism by which these cytosolic mRNAs decrease within minutes of a light-to-dark transition: active cytosolic mRNA decay, transcriptional tuning (reduced synthesis), or a defined combination of both.

  3. The plastid molecular machinery that detects reduced plastoquinone (PQH2) and activates translation of chloroplast mRNAs encoding photosynthetic complex subunits (FoF1 ATP synthase, PSII, cytochrome b6f, PSI, NDH): the identity of the PQH2-detecting machinery and the downstream effectors that modulate translation on these mRNAs.

The two auxiliary sub-questions from the same source below each stand alone: (a) the functional significance of light-dependent chlororibosome pausing on psbA mRNA (which shows interspecies variation) for photosynthetic complex assembly or protein quality control; and (b) whether extended periods of darkness induce translational repression through the (p)ppGpp stringent response in cyanobacteria. The plastid machinery sought senses reduced plastoquinone (PQH2) directly, distinct from correlated consequences of perturbing photosynthetic electron transport (energy charge, ATP/GTP levels, thylakoid proton gradient, photoreceptor signaling).

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