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SUMMARY Translation of the chloroplastpsbAmRNA in angiosperms is activated by photodamage of its gene product, the D1 subunit of photosystem II (PSII), providing nascent D1 for PSII repair. The involvement of chlorophyll in the regulatory mechanism has been suggested due to the regulatory roles of proteins proposed to mediate chlorophyll/D1 transactions and the fact that chlorophyll is synthesized only in the light in angiosperms. We used ribosome profiling and RNA‐seq to address whether the effects of light on chloroplast translation are conserved in the liverwort Marchantia (Marchantia polymorpha), which synthesizes chlorophyll in both the dark and the light. As in angiosperms, ribosome occupancy onpsbAmRNA decreased rapidly upon shifting plants to the dark and was rapidly restored upon a transfer back to the light, whereas ribosome occupancy on other chloroplast mRNAs changed very little. The results were similar in aMarchantiamutant unable to synthesize chlorophyll in the dark. Those results, in conjunction with pulse‐labeling data, suggest that light elicits a plastome‐wide activation of translation elongation and a specific increase inpsbAtranslation initiation inMarchantia, as in angiosperms. These findings show that light regulates chloroplast translation similarly in vascular and non‐vascular plants, and that constitutive chlorophyll synthesis does not affect light‐regulatedpsbAtranslation initiation. Additionally, the translational outputs of chloroplast genes are similar inMarchantiaand angiosperms but result from differing contributions of mRNA abundance and translational efficiencies. This adds to the evidence that chloroplast mRNA abundance and translational efficiencies co‐evolve under selection to maintain protein outputs.
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The High Chlorophyll Fluorescence 244 homolog CrHCF244 is required for psbA (D1) translation in Chlamydomonas reinhardtii
Abstract Translation of psbA, the chloroplast gene that encodes the D1 subunit of Photosystem II (PSII), is important for both PSII biogenesis and repair. The translation of the psbA transcript in the chloroplast is under the control of nuclear gene products. Using a Chlamydomonas forward genetic screen and whole genome sequencing, we found a mutant defective in PSII activity and mapped the causative gene to be the homolog of Arabidopsis High Chlorophyll Fluorescence 244 (HCF244) gene, or CrHCF244. We then demonstrated that CrHCF244 is required for psbA translation in the alga, consistent with the function of HCF244 in Arabidopsis. The Arabidopsis HCF244 gene also partially complemented the algal mutant. These results experimentally support the functional conservation of the homologs in green algae and land plants. Intriguingly, the CrHCF244 mutant also exhibited a relatively high rate of suppressor mutants, pointing to the presence of alternative factor(s)/pathway(s) for D1 translation control. The establishment of CrHCF244 as a psbA translation factor in Chlamydomonas showed the similarities in psbA translation regulation in algae and plants. The future identification of the alternative factor(s) in this alga will provide insights on psbA translation in plants.
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- Award ID(s):
- 1923589
- PAR ID:
- 10681758
- Publisher / Repository:
- Society for Experimental Biology (SEB) and Oxford University Press (OUP)
- Date Published:
- Journal Name:
- Journal of Experimental Botany
- ISSN:
- 0022-0957
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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The D1 subunit of photosystem II (PSII) is subject to light-induced damage. In plants, D1 photodamage activates translation of chloroplastpsbAmRNA encoding D1, providing D1 for PSII repair. Three D1 assembly factors have been implicated in the regulatory mechanism: HCF244 and RBD1 activatepsbAtranslation, whereas HCF136 repressespsbAtranslation in the dark. To clarify the regulatory circuit, we analyzedpsbAribosome occupancy in dark-adapted and illuminatedrbd1andrbd1;hcf136double mutants in Arabidopsis and in Zm-hcf244and Zm-hcf244;Zm-hcf136double mutants in maize. The results show that RBD1 is required for light-inducedpsbAtranslation but has only a small effect onpsbAribosome occupancy in the dark. RBD1 is not required forpsbAtranslation when HCF136 is absent, indicating that RBD1 activatespsbAtranslation in the light by inhibiting HCF136’s repressive effect. By contrast, HCF244 is required to recruit ribosomes topsbAmRNA in light, dark, and in the absence of HCF136. We demonstrate further that HCF244 is not required for the translational activator HCF173 to bind thepsbA5’UTR. These results show that RBD1 is central to the perception of the D1 photodamage that triggers D1 synthesis and that it activatespsbAtranslation by relieving repression by an HCF136-dependent assembly intermediate. HCF244 activates downstream of those events without impacting HCF173’s binding topsbAmRNA. The results implicate a feature of nascent D1 that is affected by both HCF136 and RBD1 as the signal that reports D1 photodamage to regulatepsbAtranslation rate as needed for PSII repair.more » « less
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Abstract Translation initiation on chloroplast psbA mRNA in plants scales with light intensity, providing its gene product, D1, as needed to replace photodamaged D1 in Photosystem II. The psbA translational activator HIGH CHLOROPHYLL FLUORESCENCE 173 (HCF173) has been hypothesized to mediate this regulation. HCF173 belongs to the short-chain dehydrogenase/reductase superfamily, associates with the psbA 5′-untranslated region (5′-UTR), and has been hypothesized to enhance translation by binding an RNA segment that would otherwise pair with and mask the ribosome binding region. To test these hypotheses, we examined whether a synthetic pentatricopeptide repeat (sPPR) protein can substitute for HCF173 when bound to the HCF173 binding site. We show that an sPPR designed to bind HCF173's footprint in the psbA 5′-UTR bound the intended site in vivo and partially substituted for HCF173 to activate psbA translation. However, sPPR-activated translation did not respond to light. These results imply that HCF173 activates translation, at least in part, by sequestering the RNA it binds to maintain an accessible ribosome binding region, and that HCF173 is also required to regulate psbA translation in response to light. Translational activation can be added to the functions that can be programmed with sPPR proteins for synthetic biology applications in chloroplasts.more » « less
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Oxygenic photosynthetic organisms use Photosystem II (PSII) to oxidize water and reduce plastoquinone. Here, we review the mechanisms by which PSII is assembled and turned over in the model green alga Chlamydomonas reinhardtii. This species has been used to make key discoveries in PSII research due to its metabolic flexibility and amenability to genetic approaches. PSII subunits originate from both nuclear and chloroplastic gene products in Chlamydomonas. Nuclear-encoded PSII subunits are transported into the chloroplast and chloroplast-encoded PSII subunits are translated by a coordinated mechanism. Active PSII dimers are built from discrete reaction center complexes in a process facilitated by assembly factors. The phosphorylation of core subunits affects supercomplex formation and localization within the thylakoid network. Proteolysis primarily targets the D1 subunit, which when replaced, allows PSII to be reactivated and completes a repair cycle. While PSII has been extensively studied using Chlamydomonas as a model species, important questions remain about its assembly and repair which are presented here.more » « less
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Abstract The D1 subunit of photosystem II is subject to photooxidative damage. Photodamaged D1 must be replaced with nascent D1 to maintain photosynthesis. In plant chloroplasts, D1 photodamage regulates D1 synthesis by modulating translation initiation on psbA mRNA encoding D1, but the underlying mechanisms are unknown. Analyses of reporter constructs in transplastomic tobacco (Nicotiana tabacum) showed that the psbA translational regulator HCF173 activates via a cis-element in the psbA 5′-UTR. However, the psbA UTRs are not sufficient to program light-regulated translation. Instead, the psbA open reading frame represses translation initiation in cis, and D1 photodamage relieves this repression. HCF173 remains bound to the psbA 5′-UTR in the dark and truncation of HCF173 prevents repression in the dark, implicating HCF173 as a mediator of repression. We propose a model that accounts for these and prior observations, which is informed by structures of the Complex I assembly factor CIA30/NDUFAF1. We posit that D1 photodamage relieves a repressive cotranslational interaction between nascent D1 and HCF173's CIA30 domain, that the photosystem II assembly factor HCF136 promotes this repressive interaction, and that these events toggle HCF173 between activating and repressive conformations on psbA mRNA. These findings elucidate a translational rheostat that optimizes photosynthesis in response to shifting light conditions.more » « less
- Free Publicly Accessible Full Text
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Accepted Manuscript
- Journal Article:
- https://doi.org/10.1093/jxb/eraf362
