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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. 

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. 

Abstract Thylakoid membranes in chloroplasts and cyanobacteria harbor the multisubunit protein complexes that catalyze the light reactions of photosynthesis. In plant chloroplasts, the thylakoid membrane system comprises a highly organized network with several subcompartments that differ in composition and morphology: grana stacks, unstacked stromal lamellae, and grana margins at the interface between stacked and unstacked regions. The localization of components of the photosynthetic apparatus among these subcompartments has been well characterized. However, less is known about the localization of proteins involved in the biogenesis and repair of the photosynthetic apparatus, the partitioning of proteins between two recently resolved components of the traditional margin fraction (refined margins and curvature), and the effects of light on these features. In this study, we analyzed the partitioning of numerous thylakoid biogenesis and repair factors among grana, curvature, refined margin, and stromal lamellae fractions ofArabidopsisthylakoid membranes, comparing the results from illuminated and dark‐adapted plants. Several proteins previously shown to localize to a margin fraction partitioned in varying ways among the resolved curvature and refined margin fractions. For example, the ALB3 insertase and FtsH protease involved in photosystem II (PSII) repair were concentrated in the refined margin fraction, whereas TAT translocon subunits and proteins involved in early steps in photosystem assembly were concentrated in the curvature fraction. By contrast, two photosystem assembly factors that facilitate late assembly steps were depleted from the curvature fraction. The enrichment of the PSII subunit OE23/PsbP in the curvature fraction set it apart from other PSII subunits, supporting the previous conjecture that OE23/PsbP assists in PSII biogenesis and/or repair. The PSII assembly factor PAM68 partitioned differently among thylakoid fractions from dark‐adapted plants and illuminated plants and was the only analyzed protein to convincingly do so. These results demonstrate an unanticipated spatial heterogeneity of photosystem biogenesis and repair functions in thylakoid membranes and reveal the curvature fraction to be a focal point of early photosystem biogenesis. 

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. 

Abstract Signals emanating from chloroplasts influence nuclear gene expression, but roles of retrograde signals during chloroplast development are unclear. To address this gap, we analyzed transcriptomes of non-photosynthetic maize mutants and compared them to transcriptomes of stages of normal leaf development. The transcriptomes of two albino mutants lacking plastid ribosomes resembled transcriptomes at very early stages of normal leaf development, whereas the transcriptomes of two chlorotic mutants with thylakoid targeting or plastid transcription defects resembled those at a slightly later stage. We identified ∼2,700 differentially expressed genes, which fall into six major categories based on the polarity and mutant-specificity of the change. Downregulated genes were generally expressed late in normal development and were enriched in photosynthesis genes, whereas upregulated genes act early and were enriched for functions in chloroplast biogenesis and cytosolic translation. We showed further that target-of-rapamycin (TOR) signaling was elevated in mutants lacking plastid ribosomes and declined in concert with plastid ribosome buildup during normal leaf development. Our results implicate three plastid signals as coordinators of photosynthetic differentiation. One signal requires plastid ribosomes and activates photosynthesis genes. A second signal reflects attainment of chloroplast maturity and represses chloroplast biogenesis genes. A third signal, the consumption of nutrients by developing chloroplasts, represses TOR, promoting termination of cell proliferation during leaf development. 

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.