High-copy transposons have been effectively exploited as mutagens in a variety of organisms. However, their utility for phenotype-driven forward genetics has hampered by the difficulty identifying specific insertions responsible phenotypes interest. We describe new method that can substantially increase throughput linking disrupted gene to known phenotype high-copy Mutator (Mu) transposon lines maize. The approach uses Illumina platform obtain sequences flanking Mu elements pooled, bar-coded...

Most life is ultimately sustained by photosynthesis and its rate-limiting carbon fixing enzyme, ribulose-1,5-bis-phosphate carboxylase/oxygenase (Rubisco). Although the structurally comparable cyanobacterial Rubisco amenable to in vitro assembly, higher plant enzyme has been refractory such manipulation due poor understanding of assembly pathway. Here, we report identification a chloroplast protein required for accumulation maize (Zea mays), RUBISCO ACCUMULATION FACTOR1 (RAF1), which lacks...

Abstract Chloroplast genomes in land plants harbor ∼20 group II introns. Genetic approaches have identified proteins involved the splicing of many these introns, but to date cannot account for large size intron ribonucleoprotein complexes and are not sufficient reconstitute vitro. Here, we describe an additional protein that promotes chloroplast vivo. This protein, RNC1, was by mass spectrometry analysis maize (Zea mays) coimmunoprecipitate with two previously factors, CAF1 CAF2. RNC1 is a...

The regulation of chloroplast translation by nuclear gene products makes a major contribution to the control expression, but underlying mechanisms are poorly understood. We describe pentatricopeptide repeat (PPR) protein in maize, ATP4, that is necessary for atpB open reading frame. demonstrate ATP4 associates vivo with sequences near 5' end unusually long UTR atpB/E mRNA, it facilitates ribosome association this and required accumulation activity ATP synthase. multifunctional, also enhances...

Summary Ribulose‐1,5‐bisphosphate carboxylase/oxygenase (Rubisco) plays a critical role in sustaining life by catalysis of carbon fixation the Calvin–Benson pathway. Incomplete knowledge assembly pathway chloroplast Rubisco has hampered efforts to fully delineate enzyme's properties, or seek improved catalytic characteristics via directed evolution. Here we report that Mu transposon insertion Zea mays (maize) gene encoding dimerization co‐factor hepatocyte nuclear factor 1 ( DC...

Abstract Chloroplasts and other members of the plastid organelle family contain a small genome bacterial ancestry. Young chloroplasts hundreds copies, but functional significance this high copy number has been unclear. We describe molecular phenotypes associated with mutations in nuclear gene maize (Zea mays), white2 (w2), encoding predicted organellar DNA polymerase. Weak strong mutant alleles cause moderate (approximately 5-fold) severe 100-fold) decrease number, respectively, as assayed...

Abstract Chloroplast transcription in land plants relies on collaboration between a plastid-encoded RNA polymerase (PEP) of cyanobacterial ancestry and nucleus-encoded phage ancestry. PEP associates with additional proteins that are unrelated to bacterial factors, many which have been shown be important for activity Arabidopsis (Arabidopsis thaliana). However, the biochemical roles these PEP-associated not known. We describe phenotypes conditioned by transposon insertions genes encoding...

Abstract The D1 subunit of photosystem II is subject to photooxidative damage. Photodamaged must be replaced with nascent maintain photosynthesis. In plant chloroplasts, photodamage regulates synthesis by modulating translation initiation on psbA mRNA encoding D1. underlying mechanisms are unknown. Analyses reporter constructs in transplastomic tobacco showed that the translational activator HCF173 activates via a cis-element 5'-UTR. However, UTRs not sufficient program light-regulated...

Abstract A chloroplast signal recognition particle (SRP) that is related to the SRP involved in secretion bacteria and eukaryotic cells used for insertion of light-harvesting chlorophyll proteins (LHCPs) into thylakoid membranes. conserved component mechanism a membrane-bound receptor, denoted FtsY bacteria. Plant genomes encode homologs are targeted (cpFtsY). To investigate vivo roles cpFtsY, we characterized maize cpFtsY mutants having Mu transposon corresponding gene (chloroplast...

Summary The expression of chloroplast genes relies on a host nucleus‐encoded proteins. Identification such proteins and elucidation their functions are ongoing challenges. We used ribosome profiling to revisit the function pentatricopeptide repeat protein LPE1, reported stimulate translation psbA mRNA in Arabidopsis. Mutation maize LPE1 ortholog causes photosystem II (PSII) deficiency defect psbJ open reading frame (ORF) but has no effect expression. To reflect this function, we named T...

Synthesis of the D1 reaction center protein Photosystem II is dynamically regulated in response to environmental and developmental cues. In chloroplasts, much this regulation occurs at post-transcriptional level, but proteins responsible are largely unknown. To discover that impact psbA expression, we identified associate with maize mRNA by: (i) formaldehyde cross-linking leaf tissue followed by antisense oligonucleotide affinity capture mRNA; (ii) co-immunoprecipitation HCF173, a...

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 non-photosynthetic maize mutants and compared them to stages normal leaf development. The two albino lacking plastid ribosomes resembled at very early development, whereas the chlorotic with thylakoid targeting or transcription defects those a slightly later stage. We identified ∼2,700 differentially...

Abstract The D1 reaction center protein of photosystem II is subject to photooxidative damage. Photodamaged must be replaced with newly synthesized maintain photosynthesis. In plant chloroplasts, synthesis coupled photodamage via regulated translation initiation on psbA mRNA, which encodes D1. Mechanisms underlying this coupling are unclear. We show by analysis reporter constructs in tobacco that the translational activators HCF173 and HCF244 activate cis -elements UTRs 5’ UTR sequence bound...

Thymidine kinase (TK) is a key enzyme of the salvage pathway that recycles thymidine nucleosides to produce deoxythymidine triphosphate. Here, we identified single TK maize (Zea mays), denoted CPTK1, as necessary in replication plastidial genome (cpDNA), demonstrating essential function during chloroplast biogenesis. CPTK1 localized both plastids and mitochondria, its absence resulted an albino phenotype, reduced cpDNA copy number severe deficiency ribosomes. Mitochondria were not affected,...

Bacterial ribosome hibernation factors sequester ribosomes in an inactive state during the stationary phase and response to stress. The cyanobacterial factor LrtA has been suggested inactivate dark be important for post-stress survival. In this study, we addressed hypothesis that Plastid Specific Ribosomal Protein 1 (PSRP1), chloroplast-localized homolog plants, contributes global repression of chloroplast translation occurs when plants are shifted from light dark. We found abundance PSRP1...

ABSTRACT The D1 reaction center protein of photosystem II (PSII) is subject to light-induced damage. In plants, photodamage activates translation the chloroplast psbA mRNA encoding D1, providing for PSII repair. Genetic data have implicated three assembly factors in regulatory mechanism: HCF244 and RBD1 activate whereas HCF136 represses dark. To clarify circuit, we analyzed ribosome occupancy dark-adapted illuminated rbd1 hcf136;rbd1 double mutants Arabidopsis, Zm- hcf244 hcf136; maize....

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 four non-photosynthetic maize mutants and interpreted them in the context transcriptome dynamics normal leaf development. We two albino lacking plastid ribosomes chlorotic with thylakoid targeting or transcription defects. The ∼2700 differentially expressed genes fall into six major...