atpF Intron Research Articles

DNA barcode screening, identification of germplasm resources, and analysis of genetic diversity of Anemarrhena asphodeloides

Anemarrhena asphodeloides is a common medicinal material used in clinical prescriptions and Chinese patent medicine. In this study, the Illumina platform was used to obtain the chloroplast genome sequences of seven kinds of A. asphodeloides from different areas. The specific DNA barcodes were screened by comparative genomics analysis, and the DNA barcodes were used to identify the germplasm resources and analyze the genetic diversity of A. asphodeloides samples from different areas in China. All the seven chloroplast genomes had a ring structure. The total length was 156 801-156 930 bp, and 113 genes were annotated, including 79 protein-coding genes, 30 tRNA genes, and four rRNA genes. The comparative genomics analysis showed that rps16, trnG-GCC, atpF, rpoB, ycf3, rpl16, ndhF, trnS-GCU＿trnG-GCC, petN-psbM, and ndhF-rpl32 were potential candidates for specific DNA barcodes of A. asphodeloides. In this study, the second intron of ycf3 and atpF intron sequences with a sequence length of 700-800 bp and easy amplification were selected for polymerase chain reaction(PCR) amplification and sequencing of 594 samples from 26 areas. The sequence analysis showed that six and eight haplotypes of ycf3 and atpF sequences could be identified, respectively, and 17 haplotypes could be identified by combined analysis of the two sequences, which were named Hap1-Hap17. The haplotype diversity(H_d), nucleotide diversity(P_i), and genetic distance of A. asphodeloides in 26 populations were 0.68, 0.93×10~(-3), and 0-0.003 1, respectively, indicating that the genetic diversity within the species of A. asphodeloides is rich. The intermediary adjacent network analysis showed that Hap5 was the oldest haplotype, which was mainly distributed in Yixian county of Baoding, Hebei province, Hequ county of Xinzhou, Shanxi province, and Xiangfen county of Linfen, Shanxi province. This study has important guiding significance for the identification of A. asphodeloides species, the protection and development of germplasm resources, and the identification of production areas, and it provides a research basis for further revealing the genetic evolution law of A. asphodeloides.

Genome Survey Sequencing of the Medical Plant Dioscorea futschauensis: Genomic Characteristics Estimation and Genomic Resources Development

Dioscorea futschauensis Uline ex R. Knuth, a perennial climbing herb belonging to the Stenophora section of the genus Dioscorea L. (Dioscoreaceae), originates from Southeast China and is highly regarded for its medicinal properties. Despite its medicinal significance, the absence of genomic information has impeded the effective utilization and conservation of this species. Here, we conducted genome survey sequencing of D. futschauensis to unveil its genomic characteristics and identify species-specific genomic markers, including whole plastome sequence, plastome-divergent hotspots, single-copy nuclear genes and polymorphic nuclear SSRs. The genome of D. futschauensis was approximated to be 1392.76 Mb in length, accompanied by a substantial heterozygosity rate of 1.75% and a repeat content of 67.43%. Comparative plastome analysis illuminated the conserved genome structure and gene content within D. futschauensis, and revealed four highly divergent hotspots (atpF intron, psbA-matK, matK -rps16, and rrn16-trnI) that could serve as molecular markers for D. futschauensis and its closely related species. Furthermore, more than 200 single-copy nuclear genes and 78 polymorphic nuclear SSRs were identified within D. futschauensis. Overall, the comprehensive genomic information and abundant genetic resources provided herein will not only facilitate extended research in the realms of population genetics, phylogenetics and conservation biology of D. futschauensis, but also hold considerable value for its cultivation and molecular breeding applications.

Comparative Plastid Genome and Phylogenomic Analyses of Potamogeton Species.

Potamogetonaceae are aquatic plants divided into six genera. The largest genus in the family is Potamogeton, which is morphologically diverse with many hybrids and polyploids. Potamogetonaceae plastomes were conserved in genome size (155,863 bp-156,669 bp), gene contents (113 genes in total, comprising 79 protein-coding genes and 30 tRNA and 4 rRNA genes), and GC content (36.5%). However, we detected a duplication of the trnH gene in the IR region of the Potamogeton crispus and P. maakianus plastomes. A comparative analysis of Alismatales indicated that the plastomes of Potamogetonaceae, Cymodaceae, and Ruppiaceae have experienced a 6-kb inversion of the rbcL-trnV region and the ndh complex has been lost in the Najas flexilis plastome. Five divergent hotspots (rps16-trnQ, atpF intron, rpoB-trnC, trnC-psbM, and ndhF-rpl32) were identified among the Potamogeton plastomes, which will be useful for species identification. Phylogenetic analyses showed that the family Potamogetonaceae is a well-defined with 100% bootstrap support and divided into two different clades, Potamogeton and Stuckenia. Compared to the nucleotide substitution rates among Alismatales, we found neutral selection in all plastid genes of Potamogeton species. Our results reveal the complete plastome sequences of Potamogeton species, and will be helpful for taxonomic identification, the elucidation of phylogenetic relationships, and the plastome structural analysis of aquatic plants.

The plastome of Arctic Oxytropis arctobia (Fabaceae) is significantly different from that of O. splendens and other related species.

Anatomical and physiological specializations for plant adaptation to harsh climates result from molecular mechanisms that can be encoded in the nucleus or organelle. In this study, the complete plastomes of an arctic species, Oxytropis arctobia Bunge (Fabaceae), and a closely related temperate species, O. splendens Douglas ex Hook., were assembled, annotated, and analyzed to identify differences that might help explain their adaptation to different environments. This is consistent with the previously sequenced O. bicolor DC. and O. glabra plastomes, O. arctobia and O. splendens plastomes both have the common features of the inverted repeat-lacking clade (IRLC), as well as atpF intron loss, which is unique to the genus. However, significant differences were observed between the plastomes of O. arctobia and O. splendens and other closely related species (Oxytropis spp. and Astragalus spp.), including a 3 kb inversion, two large insertions (>1 kb), significant modifications of the accD gene, and an overall larger size.

An Exon Skipping in CRS1 Is Associated with Perturbed Chloroplast Development in Maize.

Chloroplasts of higher plants are semi-autonomous organelles that perform photosynthesis and produce hormones and metabolites. They play crucial roles in plant growth and development. Although many seedling-lethal nuclear genes or regulators required for chloroplast development have been characterized, the understanding of chloroplast development is still limited. Using a genetic screen, we isolated a mutant named ell1, with etiolated leaves and a seedling-lethal phenotype. Analysis by BN-PAGE and transmission electron microscopy revealed drastic morphological defects of chloroplasts in ell1 mutants. Genetic mapping of the mutant gene revealed a single mutation (G-to-A) at the 5′ splice site of intron 5 in CRS1, resulting in an exon skipping in CRS1, indicating that this mutation in CRS1 is responsible for the observed phenotype, which was further confirmed by genetic analysis. The incorrectly spliced CRS1 failed to mediate the splicing of atpF intron. Moreover, the quantitative analysis suggested that ZmCRS1 may participate in chloroplast transcription to regulate the development of chloroplast. Taken together, these findings improve our understanding of the ZmCRS1 protein and shed new light on the regulation of chloroplast development in maize.

Chloroplast phylogenomics and character evolution of eastern Asian Astragalus (Leguminosae): Tackling the phylogenetic structure of the largest genus of flowering plants in Asia

Astragalus, as the largest genus of the flowering plants, is well-known for its high species richness and morphological diversity. Previous studies suggested that many of the subgenera of Astragalus are not monophyletic and the phylogenetic relationships within the genus are still poorly known. In this study, we sampled 117 accessions of Astragalus and its close relatives, covering 55 sections of the genus plus 30 outgroup taxa to recover the main clades of eastern Asian Astragalus based on sequences of the whole chloroplast genome and 65 chloroplast CDSs. Astragalus is supported to be monophyletic and it is sister to the Oxytropis + Coluteoid clade. Within Astragalus, we recovered ten clades, and the ten clades differ substantially from Bunge’s subgenera. The former segregate genus Astracantha is also monophyletic, but embedded within Astragalus s. str., supporting the merge of the spiny former genus Astracantha with Astragalus. We detected the atpF intron losses in the chloroplast genome of the Oxytropis + Coluteoid clade, i.e., the sister clade to Astragalus. Furthermore, we estimated the ancestral states of the trichome morphology and habit via the Bayesian Binary Method. The medifixed hair type is inferred to have developed at least five times and the annual habit originated at least six times. In addition, Astragalus is estimated to have originated in the mid Miocene (stem age, 16.09 Ma, 95% HPD: 12.46–20.50 Ma). The divergence times of the medifixed hair groups ranged from 4.03 to 0.87 Ma, mostly 2–1 Ma, which are correlated with the estimated phased uplifts of the Qinghai-Tibetan Plateau (QTP). We hypothesize that the uplifts of the QTP, which contributed to aridification in eastern Asia and the adjacent regions, may have accelerated the rapid speciation of Astragalus, especially the xerophilous groups (i.e. the medifixed hair groups).

Complete plastome sequences of two Neottia species and comparative analysis with other Neottieae species (Orchidaceae)

The tribe Neottieae (Orchidaceae subfam. Epidendroideae) represents an excellent potential system for understanding plastome evolution in orchids. The tribe includes about 100 species with six recognized genera and includes both chlorophyllous and achlorophyllous taxa. Previously published Neottieae plastomes have revealed a complicated history of gene loss, particularly ndh gene loss, as well as plastome structural changes in achlorophyllous taxa. However, the plastome structure of most Neottieae taxa remains unknown. Here we investigate plastome structure in two newly sequenced Neottia species (N. suzukii and N. japonica) and characterize sequence divergence among Neottieae plastomes. We sequenced and analysed the complete plastomes of three individuals of two Neottia species (two individuals of N. japonica and one individual of N. suzukii) and compared them to seven additional published Neottia plastomes. The total genome lengths of the plastomes of N. japonica and N. suzukii ranged from 156,978 to 157,326 bp. The newly sequenced plastomes possessed the typical structure and gene content of angiosperm plastomes. Phylogenetic analyses based on complete plastomes and the nuclear internal transcribed spacer (ITS) showed that N. japonica and N. suzukii form a clade, but the two accessions of N. japonica were in phylogenetically incongruent positions in ITS vs plastome trees. Among all three newly sequenced Neottia plastomes, five regions (ndhH-ndhA spacer, ndhA intron, ycf3 intron, atpF intron, and trnL-ndhB spacer) showed high nucleotide divergence values (> 0.15), and are therefore considered useful molecular markers for future phylogenetic and phylogeographic studies of Neottia.

Playing the Taxonomic Cupid: Matching Pistillate and Staminate Conspecifics in Dioecious Clusia (Clusiaceae)

Abstract—Dioecious reproduction, in which unisexual flowers are developed in separate individuals, is an uncommon characteristic that has evolved multiple times independently in angiosperms. Nearly all species in the neotropical genus Clusia are dioecious and most of them are incompletely known because only one sexual morph, either the staminate or the pistillate individual, has been described. Here we explored the variability in chloroplast regions atpF intron, matK, ndhA intron, petL-psbE, rpl16 intron, as well as the nrDNA ITS to evaluate conspecificity between staminate and pistillate individuals within the genus Clusia. Our results indicate that phylogenetic analyses using maximum likelihood and Bayesian inference based on nrDNA ITS data is a suitable approach to assess whether sexual morphs are conspecifics and to estimate species phylogenetic position within the genus. Staminate and pistillate individuals were effectively identified for Clusia araracuarae, C. lundellii, C. stenophylla, and C. uvitana. Distribution maps as well as full morphological descriptions for these species are provided along with comments on their phylogenetic relationships.

The complete chloroplast genome sequence of Oxytropis bicolor Bunge (Fabaceae)

The first complete chloroplast genome of Oxytropis bicolor Bunge is reported and characterized in this study. The whole chloroplast genome was 122,461 base pairs in length with 110 genes, including 76 protein-coding genes, 30 tRNAs, and 4 rRNAs. In addition, the atpF intron was absent. Maximum-likelihood (ML) phylogenetic analysis indicated that O. bicolor and species of Astragalus were closely related, which is congruent with previous studies.

Evaluation of six candidate DNA barcode loci for identification of five important invasive grasses in eastern Australia.

Invasive grass weeds reduce farm productivity, threaten biodiversity, and increase weed control costs. Identification of invasive grasses from native grasses has generally relied on the morphological examination of grass floral material. DNA barcoding may provide an alternative means to identify co-occurring native and invasive grasses, particularly during early growth stages when floral characters are unavailable for analysis. However, there are no universal loci available for grass barcoding. We herein evaluated the utility of six candidate loci (atpF intron, matK, ndhK-ndhC, psbE—petL, ETS and ITS) for barcode identification of several economically important invasive grass species frequently found among native grasses in eastern Australia. We evaluated these loci in 66 specimens representing five invasive grass species (Chloris gayana, Eragrostis curvula, Hyparrhenia hirta, Nassella neesiana, Nassella trichotoma) and seven native grass species. Our results indicated that, while no single locus can be universally used as a DNA barcode for distinguishing the grass species examined in this study, two plastid loci (atpF and matK) showed good distinguishing power to separate most of the taxa examined, and could be used as a dual locus to distinguish several of the invasive from the native species. Low PCR success rates were evidenced among two nuclear loci (ETS and ITS), and few species were amplified at these loci, however ETS was able to genetically distinguish the two important invasive Nassella species. Multiple loci analyses also suggested that ETS played a crucial role in allowing identification of the two Nassella species in the multiple loci combinations.

An evaluation of taxonomic concepts of the widespread plant genus Aglaia and its allies across Wallace’s Line (tribe Aglaieae, Meliaceae)

Similar to other species-rich taxa in the Indo-Australian Archipelago, taxonomy of the genus Aglaia (mahogany family, Meliaceae) remains problematic. This study aims to evaluate taxonomic concepts within Aglaia based on the largest dataset to-date. We analyzed sequences of 237 accessions of Aglaia and representatives of all other genera of the tribe Aglaieae, including nuclear ribosomal ITS, the trnL–trnF intron and intergenic spacer, the atpF intron and the petD region comprising the petB–petD spacer, the petD-5′ exon and the petD intron (all but the first from the plastid genome). Our analyses were set both in maximum likelihood and Bayesian frameworks, which (1) supported paraphyly of Aglaia and Aphanamixis; (2) demonstrated polyphyly of previously described sections for Aglaia; and (3) suggested delimitation problems with 57% of the morphologically “variable species” and all “complex species”. In general, there were more genetic entities than species described, which shows that the taxonomy of this group is more complex than has sometimes been previously assumed. For some species, morphological variation suggests the existence of more variants, subspecies or species within various taxa. Furthermore, our study detected additional phylogenetic entities that were geographically distinct, occurring on either side of Wallace’s Line but not on both sides. The delineation of these inter-specific taxa needs further investigation by taking into account the morphological variation within and between populations across the entire distribution.

A member of the Whirly family is a multifunctional RNA- and DNA-binding protein that is essential for chloroplast biogenesis.

‘Whirly’ proteins comprise a plant-specific protein family whose members have been described as DNA-binding proteins that influence nuclear transcription and telomere maintenance, and that associate with nucleoids in chloroplasts and mitochondria. We identified the maize WHY1 ortholog among proteins that coimmunoprecipitate with CRS1, which promotes the splicing of the chloroplast atpF group II intron. ZmWHY1 localizes to the chloroplast stroma and to the thylakoid membrane, to which it is tethered by DNA. Genome-wide coimmunoprecipitation assays showed that ZmWHY1 in chloroplast extract is associated with DNA from throughout the plastid genome and with a subset of plastid RNAs that includes atpF transcripts. Furthermore, ZmWHY1 binds both RNA and DNA in vitro. A severe ZmWhy1 mutant allele conditions albino seedlings lacking plastid ribosomes; these exhibit the altered plastid RNA profile characteristic of ribosome-less plastids. Hypomorphic ZmWhy1 mutants exhibit reduced atpF intron splicing and a reduced content of plastid ribosomes; aberrant 23S rRNA metabolism in these mutants suggests that a defect in the biogenesis of the large ribosomal subunit underlies the ribosome deficiency. However, these mutants contain near normal levels of chloroplast DNA and RNAs, suggesting that ZmWHY1 is not directly required for either DNA replication or for global plastid transcription.

Characterization of the Molecular Basis of Group II Intron RNA Recognition by CRS1-CRM Domains

CRM (chloroplast RNA splicing and ribosome maturation) is a recently recognized RNA-binding domain of ancient origin that has been retained in eukaryotic genomes only within the plant lineage. Whereas in bacteria CRM domains exist as single domain proteins involved in ribosome maturation, in plants they are found in a family of proteins that contain between one and four repeats. Several members of this family with multiple CRM domains have been shown to be required for the splicing of specific plastidic group II introns. Detailed biochemical analysis of one of these factors in maize, CRS1, demonstrated its high affinity and specific binding to the single group II intron whose splicing it facilitates, the plastid-encoded atpF intron RNA. Through its association with two intronic regions, CRS1 guides the folding of atpF intron RNA into its predicted "catalytically active" form. To understand how multiple CRM domains cooperate to achieve high affinity sequence-specific binding to RNA, we analyzed the RNA binding affinity and specificity associated with each individual CRM domain in CRS1; whereas CRM3 bound tightly to the RNA, CRM1 associated specifically with a unique region found within atpF intron domain I. CRM2, which demonstrated only low binding affinity, also seems to form specific interactions with regions localized to domains I, III, and IV. We further show that CRM domains share structural similarities and RNA binding characteristics with the well known RNA recognition motif domain.

The complete nucleotide sequence of the cassava (Manihot esculenta) chloroplast genome and the evolution of atpF in Malpighiales: RNA editing and multiple losses of a group II intron

The complete sequence of the chloroplast genome of cassava (Manihot esculenta, Euphorbiaceae) has been determined. The genome is 161,453 bp in length and includes a pair of inverted repeats (IR) of 26,954 bp. The genome includes 128 genes; 96 are single copy and 16 are duplicated in the IR. There are four rRNA genes and 30 distinct tRNAs, seven of which are duplicated in the IR. The infA gene is absent; expansion of IRb has duplicated 62 amino acids at the 3' end of rps19 and a number of coding regions have large insertions or deletions, including insertions within the 23S rRNA gene. There are 17 intron-containing genes in cassava, 15 of which have a single intron while two (clpP, ycf3) have two introns. The usually conserved atpF group II intron is absent and this is the first report of its loss from land plant chloroplast genomes. The phylogenetic distribution of the atpF intron loss was determined by a PCR survey of 251 taxa representing 34 families of Malpighiales and 16 taxa from closely related rosids. The atpF intron is not only missing in cassava but also from closely related Euphorbiaceae and other Malpighiales, suggesting that there have been at least seven independent losses. In cassava and all other sequenced Malphigiales, atpF gene sequences showed a strong association between C-to-T substitutions at nucleotide position 92 and the loss of the intron, suggesting that recombination between an edited mRNA and the atpF gene may be a possible mechanism for the intron loss.

CRS1, a Chloroplast Group II Intron Splicing Factor, Promotes Intron Folding through Specific Interactions with Two Intron Domains

Group II introns are ribozymes that catalyze a splicing reaction with the same chemical steps as spliceosome-mediated splicing. Many group II introns have lost the capacity to self-splice while acquiring compensatory interactions with host-derived protein cofactors. Degenerate group II introns are particularly abundant in the organellar genomes of plants, where their requirement for nuclear-encoded splicing factors provides a means for the integration of nuclear and organellar functions. We present a biochemical analysis of the interactions between a nuclear-encoded group II splicing factor and its chloroplast intron target. The maize (Zea mays) protein Chloroplast RNA Splicing 1 (CRS1) is required specifically for the splicing of the group II intron in the chloroplast atpF gene and belongs to a plant-specific protein family defined by a recently recognized RNA binding domain, the CRM domain. We show that CRS1's specificity for the atpF intron in vivo can be explained by CRS1's intrinsic RNA binding properties. CRS1 binds in vitro with high affinity and specificity to atpF intron RNA and does so through the recognition of elements in intron domains I and IV. These binding sites are not conserved in other group II introns, accounting for CRS1's intron specificity. In the absence of CRS1, the atpF intron has little uniform tertiary structure even at elevated [Mg2+]. CRS1 binding reorganizes the RNA, such that intron elements expected to be at the catalytic core become less accessible to solvent. We conclude that CRS1 promotes the folding of its group II intron target through tight and specific interactions with two peripheral intron segments.

CRS1 is a novel group II intron splicing factor that was derived from a domain of ancient origin.

Protein-dependent group II intron splicing provides a forum for exploring the roles of proteins in facilitating RNA-catalyzed reactions. The maize nuclear gene crs1 is required for the splicing of the group II intron in the chloroplast atpF gene. Here we report the molecular cloning of the crs1 gene and an initial biochemical characterization of its gene product. Several observations support the notion that CRS1 is a bona fide group II intron splicing factor. First, CRS1 is found in a ribonucleoprotein complex in the chloroplast, and cofractionation data provide evidence that this complex includes atpF intron RNA. Second, CRS1 is highly basic and includes a repeated domain with features suggestive of a novel RNA-binding domain. This domain is related to a conserved free-standing open reading frame of unknown function found in both the eubacteria and archaea. crs1 is the founding member of a gene family in plants that was derived by duplication and divergence of this primitive gene. In addition to its previously established role in atpF intron splicing, new genetic data implicate crs1 in chloroplast translation. The chloroplast splicing and translation functions of crs1 may be mediated by the distinct protein products of two crs1 mRNA forms that result from alternative splicing of the crs1 pre-mRNA.

Nuclear mutations that block group II RNA splicing in maize chloroplasts reveal several intron classes with distinct requirements for splicing factors.

To elucidate mechanisms that regulate chloroplast RNA splicing in multicellular plants, we sought nuclear mutations in maize that result in chloroplast splicing defects. Evidence is presented for two nuclear genes whose function is required for the splicing of group II introns in maize chloroplasts. A mutation in the crs1 (for chloroplast RNA splicing 1) gene blocks the splicing of only the atpF intron, whereas a mutation in the crs2 gene blocks the splicing of many chloroplast introns. In addition, a correlation was observed between the absence of plastid ribosomes and the failure to splice several chloroplast introns. Our results suggest that a chloroplast-encoded factor and a nuclear-encoded factor whose activity requires crs2 function facilitate the splicing of distinct sets of group II introns. These two genetically defined intron sets also differ with regard to intron structure: one set consists of only subgroup IIA introns and the other of only subgroup IIB introns. Therefore, it is likely that distinct splicing factors recognize subgroup-specific features of intron structure or facilitate subgroup-specific aspects of the splicing reaction. Of the 12 pre-mRNA introns in the maize chloroplast genome, only one is normally spliced in both crs2 mutants and in mutants lacking plastid ribosomes, indicating that few, if any, of the group II introns in the chloroplast genome undergo autocatalytic splicing in vivo.

The atpF group-II intron-containing gene from spinach chloroplasts is not spliced in transgenic Chlamydomonas chloroplasts.

In order to determine whether the group-II trans-splicing machinery of the chloroplast of Chlamydomonas reinhardtii can splice a heterologous group-II cis intron, the atpF gene of spinach was transferred into the chloroplast genome of C. reinhardtii using the atpX expression vector. The atpF gene contains a group-II intron which, like other higher plant chloroplast introns, does not self-splice in vitro. The chimeric transgene was expressed at high levels, based on the accumulation of the precursor; however, spliced products could not be detected by Northern blotting, or by RT-PCR coupled with Southern-blot hybridization of the amplified products with an exon-junction probe. These results indicate that the spinach atpF intron is not spliced in transgenic C. reinhardtii chloroplasts. Thus, splicing of chloroplast introns mediated by cellular factors may be species-specific; alternately, the group-II splicing machinery of C. reinhardtii is specific for trans spliced introns.

Correct splicing of a group II intron from a chimeric reporter gene transcript in tobacco plastids.

An in vivo test system was developed to study group II intron splicing in higher plant chloroplasts. The chimeric reporter gene uidA was constructed by translational fusion of an intron-containing segment of the plastid atpF gene with the coding region of a plastid uidA reporter gene. The chimeric uidA gene was inserted into the tobacco plastid genome by the biolistic transformation procedure using a plastid targeting vector. Correct intron excision was confirmed by Northern blot analysis, by sequencing amplified cDNAs and by accumulation of the encoded beta-glucuronidase (GUS), the expression of which was dependent on intron removal. Removal of the intron from the uidA mRNA is less efficient (< 50%) than from the atpF mRNA (> 90%). The efficiency of atpF mRNA splicing is not affected in the plasmid transformants indicating that inefficient splicing of the highly-expressed uidA mRNA is not due to depletion of factor(s) required for the atpF intron removal. A derivative of uidA, with a stop codon introduced into the loop of domain VI, was also tested. The mutations did not affect the splicing efficiency. The chimeric uidA splicing system will facilitate the study of structural and sequence requirements for group II intron splicing in plastids of higher plants.

Expression of the Large ATP Synthase Gene Cluster from Spinach Chloroplasts

Summary We have examined the expression of the spinach ( Spinacia oleracea ) chloroplast large ATP synthase gene cluster. This gene cluster encodes the small ribosomal subunit protein two (rps2), and four proteins of the ATP synthase protein complex: CF 0 -IV (atpI), CF 0 -III (atpH), CF 0 -I (atpF), and CF 1 -α (atpA). Experiments to specifically 5′ end label (cap) primary transcripts revealed two regions of transcription initiation, one located upstream of the rpen gene and the other upstream of atpH. Northern analysis revealed greater than thirty transcripts derived from this gene cluster, the majority of which are processing products derived from the primary transcripts. In addition, two RNA species were identified as containing the atpF intron in a lariat structure. Processing steps include splicing to remove the group II intron found in atpF, as well as endonucleolytic and exonucleolytic cleavages. An ordered series of processing steps is proposed.