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.

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.

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.

Efficient Replication of the Plastid Genome Requires an Organellar Thymidine Kinase.

Thymidine kinase (TK) is a key enzyme of the salvage pathway that recycles thymidine nucleosides to produce deoxythymidine triphosphate. Here, we identified the single TK of maize (Zea mays), denoted CPTK1, as necessary in the replication of the plastidial genome (cpDNA), demonstrating the essential function of the salvage pathway during chloroplast biogenesis. CPTK1 localized to both plastids and mitochondria, and its absence resulted in an albino phenotype, reduced cpDNA copy number and a severe deficiency in plastidial ribosomes. Mitochondria were not affected, indicating they are less reliant on the salvage pathway. Arabidopsis (Arabidopsis thaliana) TKs, TK1A and TK1B, apparently resulted from a gene duplication after the divergence of monocots and dicots. Similar but less-severe effects were observed for Arabidopsis tk1a tk1b double mutants in comparison to those in maize cptk1 TK1B was important for cpDNA replication and repair in conditions of replicative stress but had little impact on the mitochondrial phenotype. In the maize cptk1 mutant, the DNA from the small single-copy region of the plastidial genome was reduced to a greater extent than other regions, suggesting preferential abortion of replication in this region. This was accompanied by the accumulation of truncated genomes that resulted, at least in part, from unfaithful microhomology-mediated repair. These and other results suggest that the loss of normal cpDNA replication elicits the mobilization of new replication origins around the rpoB (beta subunit of plastid-encoded RNA polymerase) transcription unit and imply that increased transcription at rpoB is associated with the initiation of cpDNA replication.

Large-scale genetic analysis of chloroplast biogenesis in maize.

BACKGROUND: Chloroplast biogenesis involves a collaboration between several thousand nuclear genes and ~100 genes in the chloroplast. Many of the nuclear genes are of cyanobacterial ancestry and continue to perform their ancestral function. However, many others evolved subsequently and comprise a diverse set of proteins found specifically in photosynthetic eucaryotes. Genetic approaches have been key to the discovery of nuclear genes that participate in chloroplast biogenesis, especially those lacking close homologs outside the plant kingdom. SCOPE OF REVIEW: This article summarizes contributions from a genetic resource in maize, the Photosynthetic Mutant Library (PML). The PML collection consists of ~2000 non-photosynthetic mutants induced by Mu transposons. We include a summary of mutant phenotypes for 20 previously unstudied maize genes, including genes encoding chloroplast ribosomal proteins, a PPR protein, tRNA synthetases, proteins involved in plastid transcription, a putative ribosome assembly factor, a chaperonin 60 isoform, and a NifU-domain protein required for Photosystem I biogenesis. MAJOR CONCLUSIONS: Insertions in 94 maize genes have been linked thus far to visible and molecular phenotypes with the PML collection. The spectrum of chloroplast biogenesis genes that have been genetically characterized in maize is discussed in the context of related efforts in other organisms. This comparison shows how distinct organismal attributes facilitate the discovery of different gene classes, and reveals examples of functional divergence between monocot and dicot plants. GENERAL SIGNIFICANCE: These findings elucidate the biology of an organelle whose activities are fundamental to agriculture and the biosphere. This article is part of a Special Issue entitled: Chloroplast Biogenesis.

A protein with an inactive pterin-4a-carbinolamine dehydratase domain is required for Rubisco biogenesis in plants.

Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) plays a critical role in sustaining life by catalysis of carbon fixation in the Calvin-Benson pathway. Incomplete knowledge of the assembly pathway of chloroplast Rubisco has hampered efforts to fully delineate the enzyme's properties, or seek improved catalytic characteristics via directed evolution. Here we report that a Mu transposon insertion in the Zea mays (maize) gene encoding a chloroplast dimerization co-factor of hepatocyte nuclear factor 1 (DCoH)/pterin-4α-carbinolamine dehydratases (PCD)-like protein is the causative mutation in a seedling-lethal, Rubisco-deficient mutant named Rubisco accumulation factor 2 (raf2-1). In raf2 mutants newly synthesized Rubisco large subunit accumulates in a high-molecular weight complex, the formation of which requires a specific chaperonin 60-kDa isoform. Analogous observations had been made previously with maize mutants lacking the Rubisco biogenesis proteins RAF1 and BSD2. Chemical cross-linking of maize leaves followed by immunoprecipitation with antibodies to RAF2, RAF1 or BSD2 demonstrated co-immunoprecipitation of each with Rubisco small subunit, and to a lesser extent, co-immunoprecipitation with Rubisco large subunit. We propose that RAF2, RAF1 and BSD2 form transient complexes with the Rubisco small subunit, which in turn assembles with the large subunit as it is released from chaperonins.

A major role for the plastid-encoded RNA polymerase complex in the expression of plastid transfer RNAs.

Chloroplast transcription in land plants relies on collaboration between a plastid-encoded RNA polymerase (PEP) of cyanobacterial ancestry and a nucleus-encoded RNA polymerase of phage ancestry. PEP associates with additional proteins that are unrelated to bacterial transcription factors, many of which have been shown to be important for PEP activity in Arabidopsis (Arabidopsis thaliana). However, the biochemical roles of these PEP-associated proteins are not known. We describe phenotypes conditioned by transposon insertions in genes encoding the maize (Zea mays) orthologs of five such proteins: ZmPTAC2, ZmMurE, ZmPTAC10, ZmPTAC12, and ZmPRIN2. These mutants have similar ivory/virescent pigmentation and similar reductions in plastid ribosomes and photosynthetic complexes. RNA gel-blot and microarray hybridizations revealed numerous changes in plastid transcript populations, many of which resemble those reported for the orthologous mutants in Arabidopsis. However, unanticipated reductions in the abundance of numerous transfer RNAs (tRNAs) dominated the microarray data and were validated on RNA gel blots. The magnitude of the deficiencies for several tRNAs was similar to that of the most severely affected messenger RNAs, with the loss of trnL-UAA being particularly severe. These findings suggest that PEP and its associated proteins are critical for the robust transcription of numerous plastid tRNAs and that this function is essential for the prodigious translation of plastid-encoded proteins that is required during the installation of the photosynthetic apparatus.

Ribulose-1,5-bis-phosphate carboxylase/oxygenase accumulation factor1 is required for holoenzyme assembly in maize.

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 is amenable to in vitro assembly, the higher plant enzyme has been refractory to such manipulation due to poor understanding of its assembly pathway. Here, we report the identification of a chloroplast protein required for Rubisco accumulation in maize (Zea mays), RUBISCO ACCUMULATION FACTOR1 (RAF1), which lacks any characterized functional domains. Maize lines lacking RAF1 due to Mutator transposon insertions are Rubisco deficient and seedling lethal. Analysis of transcripts and proteins showed that Rubisco large subunit synthesis in raf1 plants is not compromised; however, newly synthesized Rubisco large subunit appears in a high molecular weight form whose accumulation requires a specific chaperonin 60 isoform. Gel filtration analysis and blue native gels showed that endogenous and recombinant RAF1 are trimeric; however, following in vivo cross-linking, RAF1 copurifies with Rubisco large subunit, suggesting that they interact weakly or transiently. RAF1 is predominantly expressed in bundle sheath chloroplasts, consistent with a Rubisco accumulation function. Our results support the hypothesis that RAF1 acts during Rubisco assembly by releasing and/or sequestering the large subunit from chaperonins early in the assembly process.

The pentatricopeptide repeat-SMR protein ATP4 promotes translation of the chloroplast atpB/E mRNA.

The regulation of chloroplast translation by nuclear gene products makes a major contribution to the control of chloroplast gene expression, but the underlying mechanisms are poorly understood. We describe a pentatricopeptide repeat (PPR) protein in maize, ATP4, that is necessary for translation of the chloroplast atpB open reading frame. We demonstrate that ATP4 associates in vivo with sequences near the 5' end of the unusually long 5' UTR of the atpB/E mRNA, that it facilitates ribosome association with this mRNA, and that it is required for accumulation and activity of the chloroplast ATP synthase. ATP4 is multifunctional, in that it also enhances atpA translation and is required for accumulation of specific processed atpF and psaJ transcripts. ATP4 belongs to a sub-class of PPR proteins that include a small MutS-related (SMR) domain. SMR domains had previously been associated primarily with DNA-related functions, but our findings imply that at least some PPR-SMR proteins can act on RNA. ATP4 is orthologous to the Arabidopsis protein SVR7, but the phenotypes of atp4 and svr7 mutants suggest that the functions of these orthologs have not been strictly conserved.

Effects of reduced chloroplast gene copy number on chloroplast gene expression in maize.

Chloroplasts and other members of the plastid organelle family contain a small genome of bacterial ancestry. Young chloroplasts contain hundreds of genome copies, but the functional significance of this high genome copy number has been unclear. We describe molecular phenotypes associated with mutations in a nuclear gene in maize (Zea mays), white2 (w2), encoding a predicted organellar DNA polymerase. Weak and strong mutant alleles cause a moderate (approximately 5-fold) and severe (approximately 100-fold) decrease in plastid DNA copy number, respectively, as assayed by quantitative PCR and Southern-blot hybridization of leaf DNA. Both alleles condition a decrease in most chloroplast RNAs, with the magnitude of the RNA deficiencies roughly paralleling that of the DNA deficiency. However, some RNAs are more sensitive to a decrease in genome copy number than others. The rpoB messenger RNA (mRNA) exhibited a unique response, accumulating to dramatically elevated levels in response to a moderate reduction in plastid DNA. Subunits of photosynthetic enzyme complexes were reduced more severely than were plastid mRNAs, possibly because of impaired translation resulting from limiting ribosomal RNA, transfer RNA, and ribosomal protein mRNA. These results indicate that chloroplast genome copy number is a limiting factor for the expression of a subset of chloroplast genes in maize. Whereas in Arabidopsis (Arabidopsis thaliana) a pair of orthologous genes function redundantly to catalyze DNA replication in both mitochondria and chloroplasts, the w2 gene is responsible for virtually all chloroplast DNA replication in maize. Mitochondrial DNA copy number was reduced approximately 2-fold in mutants harboring strong w2 alleles, suggesting that w2 also contributes to mitochondrial DNA replication.

Use of Illumina sequencing to identify transposon insertions underlying mutant phenotypes in high-copy Mutator lines of maize.

High-copy transposons have been effectively exploited as mutagens in a variety of organisms. However, their utility for phenotype-driven forward genetics has been hampered by the difficulty of identifying the specific insertions responsible for phenotypes of interest. We describe a new method that can substantially increase the throughput of linking a disrupted gene to a known phenotype in high-copy Mutator (Mu) transposon lines in maize. The approach uses the Illumina platform to obtain sequences flanking Mu elements in pooled, bar-coded DNA samples. Insertion sites are compared among individuals of suitable genotype to identify those that are linked to the mutation of interest. DNA is prepared for sequencing by mechanical shearing, adapter ligation, and selection of DNA fragments harboring Mu flanking sequences by hybridization to a biotinylated oligonucleotide corresponding to the Mu terminal inverted repeat. This method yields dense clusters of sequence reads that tile approximately 400 bp flanking each side of each heritable insertion. The utility of the approach is demonstrated by identifying the causal insertions in four genes whose disruption blocks chloroplast biogenesis at various steps: thylakoid protein targeting (cpSecE), chloroplast gene expression (polynucleotide phosphorylase and PTAC12), and prosthetic group attachment (HCF208/CCB2). This method adds to the tools available for phenotype-driven Mu tagging in maize, and could be adapted for use with other high-copy transposons. A by-product of the approach is the identification of numerous heritable insertions that are unrelated to the targeted phenotype, which can contribute to community insertion resources.

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.

A ribonuclease III domain protein functions in group II intron splicing in maize chloroplasts.

Chloroplast genomes in land plants harbor approximately 20 group II introns. Genetic approaches have identified proteins involved in the splicing of many of these introns, but the proteins identified to date cannot account for the large size of intron ribonucleoprotein complexes and are not sufficient to reconstitute splicing in vitro. Here, we describe an additional protein that promotes chloroplast group II intron splicing in vivo. This protein, RNC1, was identified by mass spectrometry analysis of maize (Zea mays) proteins that coimmunoprecipitate with two previously identified chloroplast splicing factors, CAF1 and CAF2. RNC1 is a plant-specific protein that contains two ribonuclease III (RNase III) domains, the domain that harbors the active site of RNase III and Dicer enzymes. However, several amino acids that are essential for catalysis by RNase III and Dicer are missing from the RNase III domains in RNC1. RNC1 is found in complexes with a subset of chloroplast group II introns that includes but is not limited to CAF1- and CAF2-dependent introns. The splicing of many of the introns with which it associates is disrupted in maize rnc1 insertion mutants, indicating that RNC1 facilitates splicing in vivo. Recombinant RNC1 binds both single-stranded and double-stranded RNA with no discernible sequence specificity and lacks endonuclease activity. These results suggest that RNC1 is recruited to specific introns via protein-protein interactions and that its role in splicing involves RNA binding but not RNA cleavage activity.

Group II intron splicing factors derived by diversification of an ancient RNA-binding domain.

Group II introns are ribozymes whose catalytic mechanism closely resembles that of the spliceosome. Many group II introns have lost the ability to splice autonomously as the result of an evolutionary process in which the loss of self-splicing activity was compensated by the recruitment of host-encoded protein cofactors. Genetic screens previously identified CRS1 and CRS2 as host-encoded proteins required for the splicing of group II introns in maize chloroplasts. Here, we describe two additional host-encoded group II intron splicing factors, CRS2-associated factors 1 and 2 (CAF1 and CAF2). We show that CRS2 functions in the context of intron ribonucleoprotein particles that include either CAF1 or CAF2, and that CRS2-CAF1 and CRS2-CAF2 complexes have distinct intron specificities. CAF1, CAF2 and the previously described group II intron splicing factor CRS1 are characterized by similar repeated domains, which we name here the CRM (chloroplast RNA splicing and ribosome maturation) domains. We propose that the CRM domain is an ancient RNA-binding module that has diversified to mediate specific interactions with various highly structured RNAs.

Maize mutants lacking chloroplast FtsY exhibit pleiotropic defects in the biogenesis of thylakoid membranes.

A chloroplast signal recognition particle (SRP) that is related to the SRP involved in secretion in bacteria and eukaryotic cells is used for the insertion of light-harvesting chlorophyll proteins (LHCPs) into the thylakoid membranes. A conserved component of the SRP mechanism is a membrane-bound SRP receptor, denoted FtsY in bacteria. Plant genomes encode FtsY homologs that are targeted to the chloroplast (cpFtsY). To investigate the in vivo roles of cpFtsY, we characterized maize cpFtsY and maize mutants having a Mu transposon insertion in the corresponding gene (chloroplast SRP receptor1, or csr1). Maize cpFtsY accumulates to much higher levels in leaf tissue than in roots and stems. Interestingly, it is present at similar levels in etiolated and green leaf tissue and was found to bind the prolamellar bodies of etioplasts. A null cpFtsY mutant, csr1-1, showed a substantial loss of leaf chlorophyll, whereas a "leaky" allele, csr1-3, conditioned a more moderate chlorophyll deficiency. Both alleles caused the loss of various LHCPs and the thylakoid-bound photosynthetic enzyme complexes and were seedling lethal. By contrast, levels of the membrane-bound components of the thylakoid protein transport machineries were not altered. The thylakoid membranes in csr1-1 chloroplasts were unstacked and reduced in abundance, but the prolamellar bodies in mutant etioplasts appeared normal. These results demonstrate the essentiality of cpFtsY for the biogenesis not only of the LHCPs but also for the assembly of the other membrane-bound components of the photosynthetic apparatus.