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In oxygenic photosynthesis, NADP+ acts as the final acceptor of the photosynthetic electron transport chain and receives electrons via the thylakoid membrane complex photosystem I (PSI) to synthesize NAPDH by the enzyme ferredoxin:NADP+ oxidoreductase. The NADP+/NADPH redox couple is essential for cellular metabolism and redox homeostasis. However, how the homeostasis of these two dinucleotides is integrated into chloroplast biogenesis remains largely unknown. Here, we demonstrate the important role of NADP+ supply for the biogenesis of PSI by examining the nad kinase 2 (nadk2) mutant in Arabidopsis (Arabidopsis thaliana), which demonstrates disrupted synthesis of NADP+ from NAD+ in chloroplasts. Although the nadk2 mutant is highly sensitive to light, the reaction center of photosystem II (PSII) is only mildly and likely only secondarily affected compared to the wild-type. Our studies revealed that the primary limitation of photosynthetic electron transport, even at low light intensities, occurs at PSI rather than at PSII in the nadk2 mutant. Remarkably, this primarily impairs the de novo synthesis of the two PSI core subunits PsaA and PsaB, leading to the deficiency of the PSI complex in the nadk2 mutant. This study reveals an unexpected molecular link between NADK activity and mRNA translation of psaA/B in chloroplasts that may mediate a feedback mechanism to adjust de novo biosynthesis of the PSI complex in response to a variable NADPH demand. This adjustment may be important to protect PSI from photoinhibition under conditions that favor acceptor side limitation.

Defects in two plastid K+/H+ EFFLUX ANTIPORTERs in Arabidopsis can be relieved by loss of a plasma membrane Na+/H+ exchanger, presumably by altering plant K+ transport.

The inner-envelope K+ EFFLUX ANTIPORTERS (KEA) 1 and 2 are critical for chloroplast development, ion homeostasis, and photosynthesis. However, the mechanisms by which changes in ion flux across the envelope affect organelle biogenesis remained elusive. Chloroplast development requires intricate coordination between the nuclear genome and the plastome. Many mutants compromised in plastid gene expression (PGE) display a virescent phenotype, that is delayed greening. The phenotypic appearance of Arabidopsis thaliana kea1 kea2 double mutants fulfills this criterion, yet a link to PGE has not been explored. Here, we show that a simultaneous loss of KEA1 and KEA2 results in maturation defects of the plastid ribosomal RNAs. This may be caused by secondary structure changes of rRNA transcripts and concomitant reduced binding of RNA-processing proteins, which we documented in the presence of skewed ion homeostasis in kea1 kea2. Consequently, protein synthesis and steady-state levels of plastome-encoded proteins remain low in mutants. Disturbance in PGE and other signs of plastid malfunction activate GENOMES UNCOUPLED 1-dependent retrograde signaling in kea1 kea2, resulting in a dramatic downregulation of GOLDEN2-LIKE transcription factors to halt expression of photosynthesis-associated nuclear-encoded genes (PhANGs). PhANG suppression delays the development of fully photosynthesizing kea1 kea2 chloroplasts, probably to avoid progressing photo-oxidative damage. Overall, our results reveal that KEA1/KEA2 function impacts plastid development via effects on RNA-metabolism and PGE.

Modifications in nucleic acids are present in all three domains of life. More than 170 distinct chemical modifications have been reported in cellular RNAs to date. Collectively termed as epitranscriptome, these RNA modifications are often dynamic and involve distinct regulatory proteins that install, remove, and interpret these marks in a site-specific manner. Covalent nucleotide modifications-such as methylations at diverse positions in the bases, polyuridylation, and pseudouridylation and many others impact various events in the lifecycle of an RNA such as folding, localization, processing, stability, ribosome assembly, and translational processes and are thus crucial regulators of the RNA metabolism. In plants, the nuclear/cytoplasmic epitranscriptome plays important roles in a wide range of biological processes, such as organ development, viral infection, and physiological means. Notably, recent transcriptome-wide analyses have also revealed novel dynamic modifications not only in plant nuclear/cytoplasmic RNAs related to photosynthesis but especially in chloroplast mRNAs, suggesting important and hitherto undefined regulatory steps in plastid functions and gene expression. Here we report on the latest findings of known plastid RNA modifications and highlight their relevance for the post-transcriptional regulation of chloroplast gene expression and their role in controlling plant development, stress reactions, and acclimation processes.

Pentatricopeptide repeat (PPR) proteins are helical repeat-proteins that bind RNA in a modular fashion with a sequence-specificity that can be manipulated by the use of an amino acid code. As such, PPR repeats are promising scaffolds for the design of RNA binding proteins for synthetic biology applications. However, the in vivo functional capabilities of artificial PPR proteins built from consensus PPR motifs are just starting to be explored. Here, we report in vivo functions of an artificial PPR protein, dPPRrbcL, made of consensus PPR motifs that were designed to bind a sequence near the 5’ end of rbcL transcripts in Arabidopsis chloroplasts. We used a functional complementation assay to demonstrate that this protein bound its intended RNA target with specificity in vivo and that it substituted for a natural PPR protein by stabilizing processed rbcL mRNA. We targeted a second protein of analogous design to the petL 5’ UTR, where it substituted for the native stabilizing PPR protein PGR3, albeit inefficiently. These results showed that artificial PPRs can be engineered to functionally mimic the class of native PPR proteins that serve as physical barriers against exoribonucleases.

DEAD-box RNA helicases (DBRHs) modulate RNA secondary structure, allowing RNA molecules to adopt the conformations required for interaction with their target proteins. RH50 is a chloroplast-located DBRH that colocalizes and is coexpressed with GUN1, a central factor in chloroplast-to-nucleus signaling. When combined with mutations that impair plastid gene expression (prors1-1, prpl11-1, prps1-1, prps21-1, prps17-1, and prpl24-1), rh50 and gun1 mutations evoke similar patterns of epistatic effects. These observations, together with the synergistic growth phenotype of the double mutant rh50-1 gun1-102, suggest that RH50 and GUN1 are functionally related and that this function is associated with plastid gene expression, in particular ribosome functioning. However, rh50-1 itself is not a gun mutant, although-like gun1-102-the rh50-1 mutation suppresses the down-regulation of nuclear genes for photosynthesis induced by the prors1-1 mutation. The RH50 protein comigrates with ribosomal particles, and is required for efficient translation of plastid proteins. RH50 binds to transcripts of the 23S-4.5S intergenic region and, in its absence, levels of the corresponding rRNA processing intermediate are strongly increased, implying that RH50 is required for the maturation of the 23S and 4.5S rRNAs. This inference is supported by the finding that loss of RH50 renders chloroplast protein synthesis sensitive to erythromycin and exposure to cold. Based on these results, we conclude that RH50 is a plastid rRNA maturation factor.

The plant-specific PALE CRESS (PAC) protein has previously been shown to be essential for photoautotrophic growth. Here we further investigated the molecular function of the PAC protein. PAC localizes to plastid nucleoids and forms large proteinaceous and RNA-containing megadalton complexes. It co-immunoprecipitates with a specific subset of chloroplast RNAs including psbK-psbI, ndhF, ndhD, and 23S ribosomal RNA (rRNA), as demonstrated by RNA immunoprecipitation in combination with high throughput RNA sequencing (RIP-seq) analyses. Furthermore, it co-migrates with premature 50S ribosomal particles and specifically binds to 23S rRNA invitro. This coincides with severely reduced levels of 23S rRNA in pac leading to translational deficiencies and related alterations of plastid transcript patterns and abundance similar to plants treated with the translation inhibitor lincomycin. Thus, we conclude that deficiency in plastid ribosomes accounts for the pac phenotype. Moreover, the absence or reduction of PAC levels in the corresponding mutants induces structural changes of the 23S rRNA, as demonstrated by invivo RNA structure probing. Our results indicate that PAC binds to the 23S rRNA to promote the biogenesis of the 50S subunit. Significance Statement PAC is an RNA-binding protein localized in plastid nucleoids that is associated invivo with the 23S rRNA as well as with psbK-psbI, ndhF, and ndhD mRNAs, and forms high molecular weight complexes. The pronounced impact of PAC on the biogenesis of plastid ribosomes severely influences translation, leaf coloration, plant growth and leads to seedling lethality.

A key characteristic of chloroplast gene expression is the predominance of posttranscriptional control via numerous nucleus-encoded RNA binding factors. Here, we explored the essential roles of the S1-domain-containing protein photosynthetic electron transfer B (petB)/ petD Stabilizing Factor (BSF) in the stabilization and translation of chloroplast mRNAs. BSF binds to the intergenic region of petB-petD, thereby stabilizing 3ʹ processed petB transcripts and stimulating petD translation. BSF also binds to the 5ʹ untranslated region of petA and activates its translation. BSF displayed nucleic-acid-melting activity in vitro, and its absence induces structural changes to target RNAs in vivo, suggesting that BSF functions as an RNA chaperone to remodel RNA structure. BSF physically interacts with the pentatricopeptide repeat protein Chloroplast RNA Processing 1 (AtCRP1) and the ribosomal release factor-like protein Peptide chain Release Factor 3 (PrfB3), whose established RNA ligands overlap with those of BSF. In addition, PrfB3 stimulated the RNA binding ability of BSF in vitro. We propose that BSF and PrfB3 cooperatively reduce the formation of secondary RNA structures within target mRNAs and facilitate AtCRP1 binding. The translation activation function of BSF for petD is conserved in Arabidopsis (Arabidopsis thaliana) and maize (Zea mays), but that for petA operates specifically in Arabidopsis. Our study sheds light on the mechanisms by which RNA binding proteins cooperatively regulate mRNA stability and translation in chloroplasts.

The chloroplast hosts photosynthesis and a variety of metabolic pathways that are essential for plant viability and acclimation processes. In this study, we show that the sole plastid UMP kinase (PUMPKIN) in Arabidopsis (Arabidopsis thaliana) associates specifically with the introns of the plastid transcripts trnG-UCC, trnV-UAC, petB, petD, and ndhA in vivo, as revealed by RNA immunoprecipitation coupled with deep sequencing (RIP-Seq); and that PUMPKIN can bind RNA efficiently in vitro. Analyses of target transcripts showed that PUMPKIN affects their metabolism. Null alleles and knockdowns of pumpkin were viable but clearly affected in growth, plastid translation, and photosynthetic performance. In pumpkin mutants, the levels of many plastid transcripts were reduced, while the amounts of others were increased, as revealed by RNA-Seq analysis. PUMPKIN is a homomultimeric, plastid-localized protein that forms in vivo RNA-containing megadalton-sized complexes and catalyzes the ATP-dependent conversion of UMP to UDP in vitro with properties characteristic of known essential eubacterial UMP kinases. A moonlighting function of PUMPKIN combining RNA and pyrimidine metabolism is discussed.

Transcription termination by the RNA polymerase (RNAP) is a fundamental step of gene expression that involves the release of the nascent transcript and dissociation of the RNAP from the DNA template. However, the functional importance of termination extends beyond the mere definition of the gene borders. Chloroplasts originate from cyanobacteria and possess their own gene expression system. Plastids have a unique hybrid transcription system consisting of two different types of RNAPs of dissimilar phylogenetic origin together with several additional nuclear encoded components. Although the basic components involved in chloroplast transcription have been identified, little attention has been paid to the chloroplast transcription termination. Recent identification and functional characterization of novel factors in regulating transcription termination in Arabidopsis chloroplasts via genetic and biochemical approaches have provided insights into the mechanisms and significance of transcription termination in chloroplast gene expression. This review provides an overview of the current knowledge of the transcription termination in chloroplasts.

Chloroplast ribosomes, which originated from cyanobacteria, comprise a large subunit (50S) and a small subunit (30S) containing ribosomal RNAs (rRNAs) and various ribosomal proteins. Genes for many chloroplast ribosomal proteins, as well as proteins with auxiliary roles in ribosome biogenesis or functioning, reside in the nucleus. Here, we identified Arabidopsis (Arabidopsis thaliana) CHLOROPLAST RIBOSOME ASSOCIATED (CRASS), a member of the latter class of proteins, based on the tight coexpression of its mRNA with transcripts for nucleus-encoded chloroplast ribosomal proteins. CRASS was acquired during the evolution of embryophytes and is localized to the chloroplast stroma. Loss of CRASS results in minor defects in development, photosynthetic efficiency, and chloroplast translation activity under controlled growth conditions, but these phenotypes are greatly exacerbated under stress conditions induced by the translational inhibitors lincomycin and chloramphenicol or by cold treatment. The CRASS protein comigrates with chloroplast ribosomal particles and coimmunoprecipitates with the 16S rRNA and several chloroplast ribosomal proteins, particularly the plastid ribosomal proteins of the 30S subunit (PRPS1 and PRPS5). The association of CRASS with PRPS1 and PRPS5 is independent of rRNA and is not detectable in yeast two-hybrid experiments, implying that either CRASS interacts indirectly with PRPS1 and PRPS5 via another component of the small ribosomal subunit or that it recognizes structural features of the multiprotein/rRNA particle. CRASS plays a role in the biogenesis and/or stability of the chloroplast ribosome that becomes critical under certain stressful conditions when ribosomal activity is compromised.

The seedling-lethal Arabidopsis thaliana high chlorophyll fluorescence145 (hcf145) mutation leads to reduced stability of the plastid tricistronic psaA-psaB-rps14 mRNA and photosystem I (PSI) deficiency. Here, we genetically mapped the HCF145 gene, which encodes a plant-specific, chloroplast-localized, modular protein containing two homologous domains related to the polyketide cyclase family comprising 37 annotated Arabidopsis proteins of unknown function. Two further highly conserved and previously uncharacterized tandem repeat motifs at the C terminus, herein designated the transcript binding motif repeat (TMR) domains, confer sequence-specific RNA binding capability to HCF145. Homologous TMR motifs are often found as multiple repeats in quite diverse proteins of green and red algae and in the cyanobacterium Microcoleus sp PCC 7113 with unknown function. HCF145 represents the only TMR protein found in vascular plants. Detailed analysis of hcf145 mutants in Arabidopsis and Physcomitrella patens as well as in vivo and in vitro RNA binding assays indicate that HCF145 has been recruited in embryophyta for the stabilization of the psaA-psaB-rps14 mRNA via specific binding to its 5' untranslated region. The polyketide cyclase-related motifs support association of the TMRs to the psaA RNA, presumably pointing to a regulatory role in adjusting PSI levels according to the requirements of the plant cell.

ORCID ID: 0000-0003-2973-9514 (J.M.) The chloroplast-encoded low molecular weight protein PsbN is annotated as a photosystem II (PSII) subunit. To elucidate the localization and function of PsbN, encoded on the opposite strand to the psbB gene cluster, we raised antibodies and inserted a resistance cassette into PsbN in both directions. Both homoplastomic tobacco (Nicotiana tabacum) mutants ΔpsbN-F and ΔpsbN-R show essentially the same PSII deficiencies. The mutants are extremely light sensitive and failed to recover from photoinhibition. Although synthesis of PSII proteins was not altered significantly, both mutants accumulated only ;25% of PSII proteins compared with the wild type. Assembly of PSII precomplexes occurred at normal rates, but heterodimeric PSII reaction centers (RCs) and higher order PSII assemblies were not formed efficiently in the mutants. The ΔpsbN-R mutant was complemented by allotopic expression of the PsbN gene fused to the sequence of a chloroplast transit peptide in the nuclear genome. PsbN represents a bitopic trans-membrane peptide localized in stroma lamellae with its highly conserved C terminus exposed to the stroma. Significant amounts of PsbN were already present in dark-grown seedling. Our data prove that PsbN is not a constituent subunit of PSII but is required for repair from photoinhibition and efficient assembly of the PSII RC.

The Arabidopsis endonuclease RNase E (RNE) is localized in the chloroplast and is involved in processing of plastid ribonucleic acids (RNAs). By expression of a tandem affinity purification-tagged version of the plastid RNE in the Arabidopsis rne mutant background in combination with mass spectrometry, we identified the novel vascular plant-specific and co-regulated interaction partner of RNE, designated RHON1. RHON1 is essential for photoautotrophic growth and together with RNE forms a distinct ∼800 kDa complex. Additionally, RHON1 is part of various smaller RNA-containing complexes. RIP-chip and other association studies revealed that a helix-extended-helix-structured Rho-N motif at the C-terminus of RHON1 binds to and supports processing of specific plastid RNAs. In all respects, such as plastid RNA precursor accumulation, protein pattern, increased number and decreased size of chloroplasts and defective chloroplast development, the phenotype of rhon1 knockout mutants resembles that of rne lines. This strongly suggests that RHON1 supports RNE functions presumably by conferring sequence specificity to the endonuclease.

Background: Over the past years, dust of green coffee beans has become known to be a relevant cause for occupational type I allergies. Up to now, allergy diagnostics is based on native green coffee bean extract which exhibits insufficient specificity due to interfering substances as well as batch-to-batch variations. No coffee allergen has been described on the molecular level so far. The aim of this study was to identify the first allergen of green coffee. Methods: The allergenicity of native green coffee bean extracts was analyzed by means of ImmunoCAP in sera of 17 symptomatic coffee workers. A Coffea arabica pJuFo cDNA phage display library was constructed and screened for IgE binding to coffee proteins with 2 sera from allergic coffee workers. By sequence analysis, a new coffee allergen (Cof a 1) was identified, expressed in Escherichia coli, and evaluated by Western blots. The frequency of sensitization was investigated by ELISA screening. Results: The Cof a 1 cDNA encoded a 32-kDa C. arabica class III chitinase. Serum IgE antibodies to the recombinant allergen were found in 3 out of 17 symptomatic coffee workers (18%), whereas only 2 of them reacted to the commercial allergy test. Conclusions: A class III chitinase of C. arabica was identified to be the first known coffee allergen Cof a 1. It may have a relevant potential for the specific diagnosis of coffee sensitization.

Plastid gene expression is crucial for organelle function, but the factors that control it are still largely unclear. Members of the so-called mitochondrial transcription termination factor (mTERF) family are found in metazoans and plants and regulate organellar gene expression at different levels. Arabidopsis (Arabidopsis thaliana) mTERF6 is localized in chloroplasts and mitochondria, and its knockout perturbs plastid development and results in seedling lethality. In the leaky mterf6-1 mutant, a defect in photosynthesis is associated with reduced levels of photosystem subunits, although corresponding messenger RNA levels are unaffected, whereas translational capacity and maturation of chloroplast ribosomal RNAs (rRNAs) are perturbed in mterf6-1 mutants. Bacterial one-hybrid screening, electrophoretic mobility shift assays, and coimmunoprecipitation experiments reveal a specific interaction between mTERF6 and an RNA sequence in the chloroplast isoleucine transfer RNA gene (trnI.2) located in the rRNA operon. In vitro, recombinant mTERF6 bound to its plastid DNA target site can terminate transcription. At present, it is unclear whether disturbed rRNA maturation is a primary or secondary defect. However, it is clear that mTERF6 is required for the maturation of trnI.2. This points to an additional function of mTERFs.

Although transcription termination is essential to generate functional RNAs, its underlying molecular mechanisms are still poorly understood in plastids of vascular plants. Here, we show that the RNA binding protein RHON1 participates in transcriptional termination of rbcL (encoding large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase) in Arabidopsis thaliana. Inactivation of RHON1 leads to enhanced rbcL read-through transcription and to aberrant accD (encoding β-subunit of the acetyl-CoA carboxylase) transcriptional initiation, which may result from inefficient transcription termination of rbcL. RHON1 can bind to the mRNA as well as to single-stranded DNA of rbcL, displays an RNA-dependent ATPase activity, and terminates transcription of rbcL in vitro. These results suggest that RHON1 terminates rbcL transcription using an ATP-driven mechanism similar to that of Rho of Escherichia coli. This RHON1-dependent transcription termination occurs in Arabidopsis but not in rice (Oryza sativa) and appears to reflect a fundamental difference between plastomes of dicotyledonous and monocotyledonous plants. Our results point to the importance and significance of plastid transcription termination and provide insights into its machinery in an evolutionary context.

†‡ SUMMARY Assembly of photosystem II (PSII) occurs sequentially and requires several auxiliary proteins, such as ALB3 (ALBINO3). Here, we describe the role of the Arabidopsis thaliana thylakoid membrane protein Tellurite resistance C (AtTerC) in this process. Knockout of AtTerC was previously shown to be seedling-lethal. This phenotype was rescued by expressing TerC fused C–terminally to GFP in the terc–1 background, and the resulting terc–1TerC–GFP line and an artificial miRNA-based knockdown allele (amiR-TerC) were used to analyze the TerC function. The alterations in chlorophyll fluorescence and thylakoid ultrastructure observed in amiR-TerC plants and terc–1TerC–GFP were attributed to defects in PSII. We show that this phenotype resulted from a reduction in the rate of de novo synthesis of PSII core proteins, but later steps in PSII biogenesis appeared to be less affected. Yeast two-hybrid assays showed that TerC interacts with PSII proteins. In particular, its interaction with the PSII assembly factor ALB3 has been demonstrated by co-immunoprecipitation. ALB3 is thought to assist in incorporation of CP43 into PSII via interaction with Low PSII Accumulation2 (LPA2) Low PSII Accumulation3 (LPA3). Homozygous lpa2 mutants expressing amiR-TerC displayed markedly exacerbated phenotypes, leading to seedling lethality, indicating an additive effect. We propose a model in which TerC, together with ALB3, facilitates de novo synthesis of thylakoid membrane proteins, for instance CP43, at the membrane insertion step.

Pentatricopeptide repeat (PPR) proteins are members of one of the largest nucleus-encoded protein families in plants. Here, we describe the previously uncharacterized maize (Zea mays) PPR gene, MPPR6, which was isolated from a Mutator-induced collection of maize kernel mutants by a cDNA-based forward genetic approach. Identification of a second mutant allele and cosegregation analysis confirmed correlation with the mutant phenotype. Histological investigations revealed that the mutation coincides with abnormities in the transfer cell layer, retardation of embryo development, and a considerable reduction of starch level. The function of MPPR6 is conserved across a wide phylogenetic distance as revealed by heterologous complementation of the Arabidopsis thaliana mutant in the orthologous APPR6 gene. MPPR6 appeared to be exclusively present in mitochondria. RNA coimmunoprecipitation and in vitro binding studies revealed a specific physical interaction of MPPR6 with the 5′ untranslated region of ribosomal protein S3 (rps3) mRNA. Mapping of transcript termini showed specifically extended rps3 5′ ends in the mppr6 mutant. Considerable reduction of mitochondrial translation was observed, indicating loss of RPS3 function. This is consistent with the appearance of truncated RPS3 protein lacking the N terminus in mppr6. Our results suggest that MPPR6 is directly involved in 5′ maturation and translation initiation of rps3 mRNA.