Translational activation by a synthetic PPR protein elucidates control of psbA translation in Arabidopsis chloroplasts.

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.

Correlated retrograde and developmental regulons implicate multiple retrograde signals as coordinators of chloroplast development in maize.

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.

Light-induced psbA translation in plants is triggered by photosystem II damage via an assembly-linked autoregulatory circuit.

The D1 reaction center protein of photosystem II (PSII) is subject to light-induced damage. Degradation of damaged D1 and its replacement by nascent D1 are at the heart of a PSII repair cycle, without which photosynthesis is inhibited. In mature plant chloroplasts, light stimulates the recruitment of ribosomes specifically to psbA mRNA to provide nascent D1 for PSII repair and also triggers a global increase in translation elongation rate. The light-induced signals that initiate these responses are unclear. We present action spectrum and genetic data indicating that the light-induced recruitment of ribosomes to psbA mRNA is triggered by D1 photodamage, whereas the global stimulation of translation elongation is triggered by photosynthetic electron transport. Furthermore, mutants lacking HCF136, which mediates an early step in D1 assembly, exhibit constitutively high psbA ribosome occupancy in the dark and differ in this way from mutants lacking PSII for other reasons. These results, together with the recent elucidation of a thylakoid membrane complex that functions in PSII assembly, PSII repair, and psbA translation, suggest an autoregulatory mechanism in which the light-induced degradation of D1 relieves repressive interactions between D1 and translational activators in the complex. We suggest that the presence of D1 in this complex coordinates D1 synthesis with the need for nascent D1 during both PSII biogenesis and PSII repair in plant chloroplasts.

Ribosome profiling elucidates differential gene expression in bundle sheath and mesophyll cells in maize.

The efficiencies offered by C4 photosynthesis have motivated efforts to understand its biochemical, genetic, and developmental basis. Reactions underlying C4 traits in most C4 plants are partitioned between two cell types, bundle sheath (BS), and mesophyll (M) cells. RNA-seq has been used to catalog differential gene expression in BS and M cells in maize (Zea mays) and several other C4 species. However, the contribution of translational control to maintaining the distinct proteomes of BS and M cells has not been addressed. In this study, we used ribosome profiling and RNA-seq to describe translatomes, translational efficiencies, and microRNA abundance in BS- and M-enriched fractions of maize seedling leaves. A conservative interpretation of our data revealed 182 genes exhibiting cell type-dependent differences in translational efficiency, 31 of which encode proteins with core roles in C4 photosynthesis. Our results suggest that non-AUG start codons are used preferentially in upstream open reading frames of BS cells, revealed mRNA sequence motifs that correlate with cell type-dependent translation, and identified potential translational regulators that are differentially expressed. In addition, our data expand the set of genes known to be differentially expressed in BS and M cells, including genes encoding transcription factors and microRNAs. These data add to the resources for understanding the evolutionary and developmental basis of C4 photosynthesis and for its engineering into C3 crops.

Correction: Multilevel effects of light on ribosome dynamics in chloroplasts program genome-wide and psbA-specific changes in translation.

[This corrects the article DOI: 10.1371/journal.pgen.1007555.].

Exploring the proteome associated with the mRNA encoding the D1 reaction center protein of Photosystem II in plant chloroplasts.

Synthesis of the D1 reaction center protein of Photosystem II is dynamically regulated in response to environmental and developmental cues. In chloroplasts, much of this regulation occurs at the post-transcriptional level, but the proteins responsible are largely unknown. To discover proteins that impact psbA expression, we identified proteins that associate with maize psbA mRNA by: (i) formaldehyde cross-linking of leaf tissue followed by antisense oligonucleotide affinity capture of psbA mRNA; and (ii) co-immunoprecipitation with HCF173, a psbA translational activator that is known to bind psbA mRNA. The S1 domain protein SRRP1 and two RNA Recognition Motif (RRM) domain proteins, CP33C and CP33B, were enriched with both approaches. Orthologous proteins were also among the enriched protein set in a previous study in Arabidopsis that employed a designer RNA-binding protein as a psbA RNA affinity tag. We show here that CP33B is bound to psbA mRNA in vivo, as was shown previously for CP33C and SRRP1. Immunoblot, pulse labeling, and ribosome profiling analyses of mutants lacking CP33B and/or CP33C detected some decreases in D1 protein levels under some conditions, but no change in psbA RNA abundance or translation. However, analogous experiments showed that SRRP1 represses psbA ribosome association in the dark, represses ycf1 ribosome association, and promotes accumulation of ndhC mRNA. As SRRP1 is known to harbor RNA chaperone activity, we postulate that SRRP1 mediates these effects by modulating RNA structures. The uncharacterized proteins that emerged from our analyses provide a resource for the discovery of proteins that impact the expression of psbA and other chloroplast genes.

Multilevel effects of light on ribosome dynamics in chloroplasts program genome-wide and psbA-specific changes in translation.

Plants and algae adapt to fluctuating light conditions to optimize photosynthesis, minimize photodamage, and prioritize energy investments. Changes in the translation of chloroplast mRNAs are known to contribute to these adaptations, but the scope and magnitude of these responses are unclear. To clarify the phenomenology, we used ribosome profiling to analyze chloroplast translation in maize seedlings following dark-to-light and light-to-dark shifts. The results resolved several layers of regulation. (i) The psbA mRNA exhibits a dramatic gain of ribosomes within minutes after shifting plants to the light and reverts to low ribosome occupancy within one hour in the dark, correlating with the need to replace damaged PsbA in Photosystem II. (ii) Ribosome occupancy on all other chloroplast mRNAs remains similar to that at midday even after 12 hours in the dark. (iii) Analysis of ribosome dynamics in the presence of lincomycin revealed a global decrease in the translation elongation rate shortly after shifting plants to the dark. The pausing of chloroplast ribosomes at specific sites changed very little during these light-shift regimes. A similar but less comprehensive analysis in Arabidopsis gave similar results excepting a trend toward reduced ribosome occupancy at the end of the night. Our results show that all chloroplast mRNAs except psbA maintain similar ribosome occupancy following short-term light shifts, but are nonetheless translated at higher rates in the light due to a plastome-wide increase in elongation rate. A light-induced recruitment of ribosomes to psbA mRNA is superimposed on this global response, producing a rapid and massive increase in PsbA synthesis. These findings highlight the unique translational response of psbA in mature chloroplasts, clarify which steps in psbA translation are light-regulated in the context of Photosystem II repair, and provide a foundation on which to explore mechanisms underlying the psbA-specific and global effects of light on chloroplast translation.

The Arabidopsis pentatricopeptide repeat protein LPE1 and its maize ortholog are required for translation of the chloroplast psbJ RNA.

The expression of chloroplast genes relies on a host of nucleus-encoded proteins. Identification of such proteins and elucidation of their functions are ongoing challenges. We used ribosome profiling to revisit the function of the pentatricopeptide repeat protein LPE1, reported to stimulate translation of the chloroplast psbA mRNA in Arabidopsis. Mutation of the maize LPE1 ortholog causes a photosystem II (PSII) deficiency and a defect in translation of the chloroplast psbJ open reading frame (ORF) but has no effect on psbA expression. To reflect this function, we named the maize LPE1 ortholog Translation of psbJ 1 (TPJ1). Arabidopsis lpe1 mutants likewise exhibit a loss of psbJ translation, and have, in addition, a decrease in psbN translation. We detected a small decrease in ribosome occupancy on the psbA mRNA in Arabidopsis lpe1 mutants, but ribosome profiling analyses of other PSII mutants (hcf107 and hcf173) in conjunction with in vitro RNA binding data strongly suggest that this is a secondary effect of their PSII deficiency. We conclude that maize TPJ1 promotes PSII synthesis by activating translation of the psbJ ORF, that this function is conserved in Arabidopsis LPE1, and that an additional role for LPE1 in psbN translation contributes to the PSII deficiency in lpe1 mutants.

Dynamics of Chloroplast Translation during Chloroplast Differentiation in Maize.

Chloroplast genomes in land plants contain approximately 100 genes, the majority of which reside in polycistronic transcription units derived from cyanobacterial operons. The expression of chloroplast genes is integrated into developmental programs underlying the differentiation of photosynthetic cells from non-photosynthetic progenitors. In C4 plants, the partitioning of photosynthesis between two cell types, bundle sheath and mesophyll, adds an additional layer of complexity. We used ribosome profiling and RNA-seq to generate a comprehensive description of chloroplast gene expression at four stages of chloroplast differentiation, as displayed along the maize seedling leaf blade. The rate of protein output of most genes increases early in development and declines once the photosynthetic apparatus is mature. The developmental dynamics of protein output fall into several patterns. Programmed changes in mRNA abundance make a strong contribution to the developmental shifts in protein output, but output is further adjusted by changes in translational efficiency. RNAs with prioritized translation early in development are largely involved in chloroplast gene expression, whereas those with prioritized translation in photosynthetic tissues are generally involved in photosynthesis. Differential gene expression in bundle sheath and mesophyll chloroplasts results primarily from differences in mRNA abundance, but differences in translational efficiency amplify mRNA-level effects in some instances. In most cases, rates of protein output approximate steady-state protein stoichiometries, implying a limited role for proteolysis in eliminating unassembled or damaged proteins under non-stress conditions. Tuned protein output results from gene-specific trade-offs between translational efficiency and mRNA abundance, both of which span a large dynamic range. Analysis of ribosome footprints at sites of RNA editing showed that the chloroplast translation machinery does not generally discriminate between edited and unedited RNAs. However, editing of ACG to AUG at the rpl2 start codon is essential for translation initiation, demonstrating that ACG does not serve as a start codon in maize chloroplasts.

Non-native, N-terminal Hsp70 molecular motor recognition elements in transit peptides support plastid protein translocation.

Previously, we identified the N-terminal domain of transit peptides (TPs) as a major determinant for the translocation step in plastid protein import. Analysis of Arabidopsis TP dataset revealed that this domain has two overlapping characteristics, highly uncharged and Hsp70-interacting. To investigate these two properties, we replaced the N-terminal domains of the TP of the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase and its reverse peptide with a series of unrelated peptides whose affinities to the chloroplast stromal Hsp70 have been determined. Bioinformatic analysis indicated that eight out of nine peptides in this series are not similar to the TP N terminus. Using in vivo and in vitro protein import assays, the majority of the precursors containing Hsp70-binding elements were targeted to plastids, whereas none of the chimeric precursors lacking an N-terminal Hsp70-binding element were targeted to the plastids. Moreover, a pulse-chase assay showed that two chimeric precursors with the most uncharged peptides failed to translocate into the stroma. The ability of multiple unrelated Hsp70-binding elements to support protein import verified that the majority of TPs utilize an N-terminal Hsp70-binding domain during translocation and expand the mechanistic view of the import process. This work also indicates that synthetic biology may be utilized to create de novo TPs that exceed the targeting activity of naturally occurring sequences.

Differential transit peptide recognition during preprotein binding and translocation into flowering plant plastids.

Despite the availability of thousands of transit peptide (TP) primary sequences, the structural and/or physicochemical properties that determine TP recognition by components of the chloroplast translocon are not well understood. By combining a series of in vitro and in vivo experiments, we reveal that TP recognition is determined by sequence-independent interactions and vectorial-specific recognition domains. Using both native and reversed TPs for two well-studied precursors, small subunit of ribulose-1,5-bis-phosphate carboxylase/oxygenase, and ferredoxin, we exposed these two modes of recognition. Toc34 receptor (34-kD subunit of the translocon of the outer envelope) recognition in vitro, preprotein binding in organellar, precursor binding in vivo, and the recognition of TPs by the major stromal molecular motor Hsp70 are specific for the physicochemical properties of the TP. However, translocation in organellar and in vivo demonstrates strong specificity to recognition domain organization. This organization specificity correlates with the N-terminal placement of a strong Hsp70 recognition element. These results are discussed in light of how individual translocon components sequentially interact with the precursor during binding and translocation and helps explain the apparent lack of sequence conservation in chloroplast TPs.

Exploring the Link between Photosystem II Assembly and Translation of the Chloroplast psbA mRNA.

Photosystem II (PSII) in chloroplasts and cyanobacteria contains approximately fifteen core proteins, which organize numerous pigments and prosthetic groups that mediate the light-driven water-splitting activity that drives oxygenic photosynthesis. The PSII reaction center protein D1 is subject to photodamage, whose repair requires degradation of damaged D1 and its replacement with nascent D1. Mechanisms that couple D1 synthesis with PSII assembly and repair are poorly understood. We address this question by using ribosome profiling to analyze the translation of chloroplast mRNAs in maize and Arabidopsis mutants with defects in PSII assembly. We found that OHP1, OHP2, and HCF244, which comprise a recently elucidated complex involved in PSII assembly and repair, are each required for the recruitment of ribosomes to psbA mRNA, which encodes D1. By contrast, HCF136, which acts upstream of the OHP1/OHP2/HCF244 complex during PSII assembly, does not have this effect. The fact that the OHP1/OHP2/HCF244 complex brings D1 into proximity with three proteins with dual roles in PSII assembly and psbA ribosome recruitment suggests that this complex is the hub of a translational autoregulatory mechanism that coordinates D1 synthesis with need for nascent D1 during PSII biogenesis and repair.

Functional Analysis of PSRP1, the Chloroplast Homolog of a Cyanobacterial Ribosome Hibernation Factor.

Bacterial ribosome hibernation factors sequester ribosomes in an inactive state during the stationary phase and in response to stress. The cyanobacterial ribosome hibernation factor LrtA has been suggested to inactivate ribosomes in the dark and to be important for post-stress survival. In this study, we addressed the hypothesis that Plastid Specific Ribosomal Protein 1 (PSRP1), the chloroplast-localized LrtA homolog in plants, contributes to the global repression of chloroplast translation that occurs when plants are shifted from light to dark. We found that the abundance of PSRP1 and its association with ribosomes were similar in the light and the dark. Maize mutants lacking PSRP1 were phenotypically normal under standard laboratory growth conditions. Furthermore, the absence of PSRP1 did not alter the distribution of chloroplast ribosomes among monosomes and polysomes in the light or in the dark, and did not affect the light-regulated synthesis of the chloroplast psbA gene product. These results suggest that PSRP1 does not play a significant role in the regulation of chloroplast translation by light. As such, the physiological driving force for the retention of PSRP1 during chloroplast evolution remains unclear.

Ribosome Profiling in Maize.

Ribosome profiling (also known as Ribo-seq) provides a genome-wide, high-resolution, and quantitative accounting of mRNA segments that are occupied by ribosomes in vivo. The method has been used to address numerous questions in bacteria, yeast, and metazoa, but its application to questions in plant biology is just beginning. This chapter provides a detailed protocol for profiling ribosomes in plant leaf tissue. The method was developed and optimized with maize, but it has been used successfully with Arabidopsis and tobacco as well. The method captures ribosome footprints from the chloroplast and cytosol in the same preparation, but it is not optimal for detecting the footprints of mitochondrial ribosomes. The protocol is robust and simpler than many of the methods reported previously for ribosome profiling in plants.

Translation and Co-translational Membrane Engagement of Plastid-encoded Chlorophyll-binding Proteins Are Not Influenced by Chlorophyll Availability in Maize.

Chlorophyll is an indispensable constituent of the photosynthetic machinery in green organisms. Bound by apoproteins of photosystems I and II, chlorophyll performs light-harvesting and charge separation. Due to the phototoxic nature of free chlorophyll and its precursors, chlorophyll synthesis is regulated to comply with the availability of nascent chlorophyll-binding apoproteins. Conversely, the synthesis and co-translational insertion of such proteins into the thylakoid membrane have been suggested to be influenced by chlorophyll availability. In this study, we addressed these hypotheses by using ribosome profiling to examine the synthesis and membrane targeting of chlorophyll-binding apoproteins in chlorophyll-deficient chlH maize mutants (Zm-chlH). ChlH encodes the H subunit of the magnesium chelatase (also known as GUN5), which catalyzes the first committed step in chlorophyll synthesis. Our results show that the number and distribution of ribosomes on plastid mRNAs encoding chlorophyll-binding apoproteins are not substantially altered in Zm-chlH mutants, suggesting that chlorophyll has no impact on ribosome dynamics. Additionally, a Zm-chlH mutation does not change the amino acid position at which nascent chlorophyll-binding apoproteins engage the thylakoid membrane, nor the efficiency with which membrane-engagement occurs. Together, these results provide evidence that chlorophyll availability does not selectively activate the translation of plastid mRNAs encoding chlorophyll apoproteins. Our results imply that co- or post-translational proteolysis of apoproteins is the primary mechanism that adjusts apoprotein abundance to chlorophyll availability in plants.

Functional Analysis of Semi-conserved Transit Peptide Motifs and Mechanistic Implications in Precursor Targeting and Recognition.

Over 95% of plastid proteins are nuclear-encoded as their precursors containing an N-terminal extension known as the transit peptide (TP). Although highly variable, TPs direct the precursors through a conserved, posttranslational mechanism involving translocons in the outer (TOC) and inner envelope (TOC). The organelle import specificity is mediated by one or more components of the Toc complex. However, the high TP diversity creates a paradox on how the sequences can be specifically recognized. An emerging model of TP design is that they contain multiple loosely conserved motifs that are recognized at different steps in the targeting and transport process. Bioinformatics has demonstrated that many TPs contain semi-conserved physicochemical motifs, termed FGLK. In order to characterize FGLK motifs in TP recognition and import, we have analyzed two well-studied TPs from the precursor of RuBisCO small subunit (SStp) and ferredoxin (Fdtp). Both SStp and Fdtp contain two FGLK motifs. Analysis of large set mutations (∼85) in these two motifs using in vitro, in organello, and in vivo approaches support a model in which the FGLK domains mediate interaction with TOC34 and possibly other TOC components. In vivo import analysis suggests that multiple FGLK motifs are functionally redundant. Furthermore, we discuss how FGLK motifs are required for efficient precursor protein import and how these elements may permit a convergent function of this highly variable class of targeting sequences.

Nano-scale characterization of the dynamics of the chloroplast Toc translocon.

Translocons are macromolecular nano-scale machines that facilitate the selective translocation of proteins across membranes. Although common in function, different translocons have evolved diverse molecular mechanisms for protein translocation. Subcellular organelles of endosymbiotic origin such as the chloroplast and mitochondria had to evolve/acquire translocons capable of importing proteins whose genes were transferred to the host genome. These gene products are expressed on cytosolic ribosomes as precursor proteins and targeted back to the organelle by an N-terminal extension called the transit peptide or presequence. In chloroplasts the transit peptide is specifically recognized by the Translocon of the Outer Chloroplast membrane (Toc) which is composed of receptor GTPases that potentially function as gate-like switches, where GTP binding and hydrolysis somehow facilitate preprotein binding and translocation. Compared to other translocons, the dynamics of the Toc translocon are probably more complex and certainly less understood. We have developed biochemical/biophysical, imaging, and computational techniques to probe the dynamics of the Toc translocon at the nanoscale. In this chapter we provide detailed protocols for kinetic and binding analysis of precursor interactions in organeller, measurement of the activity and nucleotide binding of the Toc GTPases, native electrophoretic analysis of the assembly/organization of the Toc complex, visualization of the distribution and mobility of Toc apparatus on the surface of chloroplasts, and conclude with the identification and molecular modeling Toc75 POTRA domains. With these new methodologies we discuss future directions of the field.