Plastid DNA is not detectable in the male gametes and pollen tubes of an angiosperm (Antirrhinum majus) that is maternal for plastid inheritance

Summary Prior cytological observations using DAPI/epifluorescence microscopy have suggested that the method could be used to rapidly screen plant species for their potential mode of plastid DNA transmission. Cytoplasmic DAPI-DNA aggregates were observed in generative cells of germinated pollen of Medicago sativa (alfalfa), a species known genetically to display biparental transmission, but not in Antirrhinum majus (snapdragon), a species known to be maternal for plastid transmission. If, as suggested, these aggregates are plastid DNA nucleoids, then M. sativa pollen should contain plastid DNA detectable by molecular biology methods and A. majus pollen should not. Total DNA was isolated from germinated pollen and analyzed by Southern blot hybridization. A clone containing part of the rbcL gene from the garden pea plastome was used as a probe for plastid DNA. This probe hybridized with a restriction fragment from M. sativa pollen DNA, but not detectably with A. majus pollen DNA, thereby corroborating the identification of the cytoplasmic DAPI-DNA aggregates in M. sativa pollen as plastid DNA, and confirming the cytologically determined absence of plastic DNA in A. majus pollen.     

Plastid inheritance in Oenothera: organelle genome modifies the extent of biparental plastid transmission

Summary The transmission abilities of four out of the five major plastome types of Oenothera (I–V) were analyzed in a constant nuclear background by assessing both the frequency of biparental inheritance and the extent of variegation in the progeny. Reciprocal crosses were performed between plants carrying one of four wild-type plastomes and plants carrying one of seven white plastid mutants. The frequency of biparental plastid transmission ranged from 0 to 56% depending on the plastid types involved in the crosses. The transmission abilities of the four representative wild-type plastids appear to be in the order of I > III > II > IV in the nuclear background of O. hookeri str. Johansen. In general, variegated seedlings from crosses that produced a higher frequency of biparental plastid transmission also had an increased abundance of tissue containing plastids of paternal origin. Although the transmission abilities of most Oenothera plastid mutants are comparable to the wild-type plastids, three mutant plastids derived from species having different type I plastids show three distinguishable transmission patterns. This study confirms the significant role of the plastome in the process of plastid transmission and possibly in plastid multiplication. However, the hypothesis of differential plastid multiplication rates suggested by earlier studies can explain the results only partially. The initiation of plastid multiplication within the newly formed zygote also seems to be plastome-dependent.     

Cell type determines plastid transmission in tomato intergeneric somatic hybrids

Summary Mesophyll (M)- and suspension culture (S)-derived protoplasts of both Lycopersicon esculentum, tomato, and its wild relative Solanum lycopersicoides were fused as S+M, M+M and S+S combinations, respectively, to resolve the role of parental cell types in determining cpDNA transmission to intergeneric somatic hybrid plants. The mesophyll cpDNA was preferentially transmitted to 96% of the plants, each regenerated from a separate callus, in M+S and S+M fusion combinations. In contrast, for the M+M combination there was an equable distribution of either tomato cpDNA or that of S. lycopersicoides among the 34 hybrid plants. The number of plastids or proplastids in mesophyll or suspension protoplasts was not a factor regulating cpDNA transmission. Mesophyll or suspension protoplasts of both fusion partners had comparable frequencies of either plastid type with a mean of 23. The biased transmission of plastids from the mesophyll parent in somatic hybrid plants of S+M and M+S combinations appears to be due to differential multiplication of plastids, possibly conditioned by an unequal input of the nucleoids found in plastids versus proplastids. In the M+M fusion, plastid and nucleotid input and subsequent plastid multiplication are apparently equal, and when combined with random sorting out leads to an equal distribution of parental cpDNAs in the regenerated somatic hybrid plants. For the S+S combination, 22 somatic hybrid plants have exclusively tomato cpDNA, an outcome that is not readily explained by donor cell input.     

Cytological demonstration of chloroplast DNA behavior during gametogenesis and zygote formation in Chlamydomonas reinhardtii

Summary We used the flourescent dye DAPI to visualize nucleoids of chloroplast DNA and follow their behavior through sexual reproduction by counting nucleoids in fixed cells at various stages. Nucleoid number varied greatly among cells at each stage. The mean number of nucleoids per cell was similar in mt ⁺ and mt ^– vegetative cells, and declined similarly during gametogenesis. Longer periods of nitrogen starvation reduced the mean nucleoid number further. Mean nucleoid number declined again in mating pairs, and continued to drop in zygotes up to the latest stage that can be examined (24-h zygotes). The oldest zygotes had means of about 2 to 3 nucleoids in different experiments, significantly fewer than in the mt ⁺ gametes (usually 4 to 5). The quantitative data on nucleoid number, mating efficiency, and germination efficiency allowed us to show that the decrease in nucleoid number is not limited to gametes that do not mate, or to zygotes that do not germinate. These data are consistant with earlier biochemical studies showing loss of chloroplast DNA during gametogenesis in both mating types, and with the degradation of paternal chloroplast DNA detected biochemically and (in non-quantitative studies) by DAPI staining. There may also be some fusion of nucleoids, although if it occurs it is not complete by 24 h of zygote maturation.     

Control of organelle transmission in Chlorophytum

Summary In a study of pollen ultrastructure in Chlorophytum comosum plastids were found to be present and apparently unaltered in both vegetative and generative cells. These ultrastructural data are consistent with the biparental transmission of plastids in this genus. However, mitochondria appear degenerate when compared to leaf mitochondria, exhibiting small myelin-like figures in addition to an abnormal cristae system. Since the plastids are not degenerate in the pollen of this species it appears that the transmission genetics of the mitochondria are determined separately from that of the plastid in higher plants.     

Non-random plastid segregation in somatic hybrids of Datura innoxia with several other Solanaceous species

Summary A non-random plastid segregation was found in somatic hybrids of Datura innoxia with seven different Solanaceous species. 14 out of 17 examined somatic hybrids showed the plastid features of Datura innoxia. Within the limits of sensitivity of the applied methods, one line could be shown to contain mixed plastids. Since sexual offspring of this line contains only one set of plastids, it is assumed that this is probably a periclinal chimaera due to the plastome, i.e., the plastid mixture is present on a plant rather than a cell level.     

Plastid inheritance in Oenothera: paternal input may influence transmission patterns

Summary Prior genetic analysis of Oenothera to assess the mechanism(s) controlling differential (biparental) plastid transmission patterns have indicated that the plastome plays an integral role. However, the influence of putative variation in paternal plastid input remains unclear. Pollen collected from Oenothera hookeri plants containing one of four different plastome types (I–IV) in a constant nuclear background (A₁A₁) was examined cytologically by DAPI/ epifluorescence microscopy. The number of plastid DNA aggregates per pollen generative cell was found to differ significantly. Plants containing plastome types I or II displayed an average of about ten plastid DNA aggregates per generative cell whereas plants containing types III or IV displayed, on average, 15 plastid DNA aggregates. The potential paternal plastid contribution to the egg cell at syngamy (III=IV>I=II) differs from the previously determined survival frequencies of the same four plastid types (I>III>II>IV) progeny.Scientific article no. A-5036, contribution no. 8084 of the Maryland Agricultural Experimental Station     

Cultivar variability for the presence of plastid DNA in pollen of Pisum sativum L.: implications for plastid transmission

Summary The mode of plastid transmission in the garden pea (Pisum sativum L.) was analyzed cytologically using the DNA-fluorochrome 4,6-diamidino-2-phenylindole (DAPI) in conjunction with epifluorescence microscopy. The reproductive cells of mature pollen obtained from 12 inbred lines and cv Early Alaska were examined for the presence or absence of DAPI-stained plastid DNA aggregates. Plastid DNA was detected in all 13 pea lines examined, although there was variability with regard to the percentage of pollen graines showing plastid DNA aggregates of generative cells (ranging from 3% in accession 82-12r to 65% in accession 82-14n). These cytological results may indicate genetic variability for plastic DNA inheritance in the garden pea. This paper is dedicated to the memory of Gerald A. Marx     

PCR analysis of pulsed-field gel electrophoresis-purified plastid DNA,a sensitive tool to judge the hetero-/homoplastomic status of plastid transformants

The genetic transformation of plastids of higher plants has developed into a powerful approach for both basic research and biotechnology. Due to the high copy number of the plastid genome per plastid and per cell, repeated cycles of shoot regeneration under conditions selective for the modified plastid chromosome are required to obtain transformants entirely lacking wild-type plastid genomes. The presence of promiscuous plastid DNA in nuclear and/or mitochondrial genomes that generally contaminate even gradient-purified plastid fractions reduces the applicability of the highly sensitive PCR approach to monitor the absence of residual wild-type plastid chromosomes in transformed lines. It is therefore difficult, or even impossible, to assess reliably the hetero- or homoplastomic state of plastid transformants in this manner. By analysing wild-type and transplastomic mutants of tobacco, we demonstrate that separation of plastid chromosomes isolated from gradient-purified plastid fractions by pulsed-field gel electrophoresis can overcome the problem of (co)amplification of interfering promiscuous plastid DNA. PCR analyses with primers specific for plastid, mitochondrial and nuclear genes reveal an impressive purity of such plastid DNA fractions at a detection limit of less than one wild-type plastid chromosome copy per ten transplastomic cells.     

Light and benzylaminopurine induce changes in ultrastructure and gene expression in plastids of Petunia hybrida cell cultures

Summary The regulatory effect of light and the cytokinin 6-benzylaminopurine (BA) on the plastid ultrastructure and plastid DNA gene expression is studied in white and mutant green cell suspension cultures of Petunia hybrida. By electron microscopy we show that both light and 6-benzylaminopurine induce the formation of thylakoid membranes and grana structures in plastids of the green cultures. For membrane formation in plastids of white cultures, light in combination with BA is required. Light and benzylaminopurine also influence the plastid DNA gene expression. By in-organello protein synthesis with isolated plastids we show that light as well as benzylaminopurine affects the synthesis of plastid DNA encoded proteins. A characteristic effect of benzylaminopurine on plastids from white and green cultures is the reduction in the synthesis of the CFI subunits of 55,000 and 57,000 D, and the reduction in the synthesis of large polypeptides with a molecular weight higher than 67,000 D. In contrast to benzylaminopurine, light only affects the DNA gene expression of plastids from white cell cultures, that are in a very early stage of plastid development. Light stimulates the synthesis of polypeptides with a molecular weight of 84,000, 70,000 and 46,000 D which are encoded by cpDNA in these white culture plastids. In green cell cultures both plastids with a etioplast-like phenotype and with a chloroplast like morphology synthesize similar polypeptides, resulting in the same polypeptide pattern. Our results indicate that qualitative differences in plastid DNA gene expression as an effect of light do occur but only in plastids at very early stages of chloroplast development. We observe a gradual reduction in the number of high molecular weight polypeptides at later stages of chloroplast development. This suggests that these large polypeptides are characteristic for plastids at an early developmental stage.Abbreviations LSU of RuBPCase large subunit of Ribulose-1, 5-bisphosphate carboxylase - CF₁ coupling factor of the ATPase complex - LCH chlorophyll a/b protein - BA 6-benzylaminopurine - cpDNA chloroplast DNA     

Loss of mt +-derived zygotic chloropiast DNA is associated with a lethal allele in Chlamydomonas monoica

Summary The Chlamydomonas monoica mutant allele mtl-1, is associated with the formation of nonviable zygospores following self-mating of the mutant strain. Furthermore, mtl-1 heterozygote populations show a 50% reduction in germination frequency and no transmission of a chloroplast antibiotic resistance marker carried by the mtl-1 parent. To determine whether the effects on zygospore viability and chloroplast gene transmission resulted from the direct involvement of the mtl-1 locus in the control of mt ⁺-directed uniparental inheritance of chloroplast DNA (cpDNA), we have used the DNA-specific fluorochrome DAPI to follow the fate of cpDNA during the maturation of zygotes. Throughout the first few hours after the initial fusion of gametes, the young zygotes show DAPI-fluorescent nucleoids distributed symmetrically around the region of nuclear fusion, and presumably located within both of the parental chloroplasts. Wild-type and mtl-1 mutant zygotes show similar early staining patterns. As the zygotes age, the staining patterns become asymmetric for the wildtype population, with all of the visible cytoplasmic nucleoids restricted to one side of the zygote. In contrast, mtl-1 homozygotes appear to lose cytoplasmic nucleoids from both sides of the zygote simultaneously and within 24 h are apparently devoid of cpDNA. By introducing a mutation which arrests cell fusion (and prevents plastid fusion), we can show that (1) the asymmetric nucleoid distribution in wildtype zygotes results from the loss of nucleoids from one gamete in each mating event, and (2) the additional loss of cpDNA in mtl-1 homozygotes does not require contact between parental plastids (thus the nuclease responsible for cpDNA degradation is not sequestered within the chloroplast of one gamete). We propose that the mtl-1 mutant strain is defective for a process which normally protects cpDNA of mt ⁺ origin.     

A model for the rapid vegetative segregation of multiple chloroplast genomes in Chlamydomonas: Assumptions and predictions of the model

Summary Physical evidence indicates that the chloroplast DNA of Chlamydomonas reinhardtii is composed of approximately 75 copies of a small unique sequence. Genetic analysis of zygotes biparental for chloroplast genes shows rapid vegetative segregation of parental chloroplast alleles. Zygote clones composed entirely of homoplasmic progeny cells predominate within 10–20 post-mating generations. A model is proposed here which reconciles the high multiplicity of chloroplast genes with their rapid vegetative segregation rates. Clustering of genomes into a small number of discrete areas (nucleoids) within the chloroplast reduces the effective number of segregating units. A non-random distribution of nucleoids to daughter cells, dictated solely by the spatial arrangement of parental nucleoids with respect to the plane of chloroplast division, further increases the rate of segregation from heteroplasmic cells. Recombination between parental chloroplast genomes is viewed as an indication of nucleoid fusion, and can account for differences in the patterns and rates of segregation at different gene loci. Within such fused nucleoids, clustering of parental genomes and a non-random distribution, again based solely on physical positioning of the genomes, to daughter nucleoids, could act to promote rapid genetic purification of heteroplasmic nucleoids. The effects of biased parental nucleoid ratios, and of potentially unequal nucleoid distributions to daughter chloroplasts are also discussed with respect to observed rates and patterns of chloroplast gene segregation.     

rps10, unreported for plastid DNAs,is located on the cyanelle genome of Cyanophora paradoxa and is contranscribed with the str operon genes

Summary rps10, encoding the plastid ribosomal protein S10, is a nuclear gene in higher plants and green algae, and is missing from the large ribosomal protein gene cluster of chlorophyll b-type plastids that contains components of the prokaryotic S10, spc and alpha operons. The cyanelle genome of Cyanophora paradoxa is shown to harbor rps10 as another specific feature of its organization. However, this novel plastid gene is not contiguous with the genes of the S10 operon, but is adjacent to, and cotranscribed with, the str operon, a trait also found in archaebacteria.     

Phylogenetic analysis of plastid origins based on secA sequences

We have generated secA sequence data from a number of photosynthetic prokaryotes and carried out a phylogenetic analysis using secA sequences from prokaryotes, green plants, and red and brown algae. We have studied the substitution patterns that give rise to the apparent phylogenetic structure. We show that the high AT content of the plastid sequences significantly affects the amino-acid composition. We also show that most of the apparent evidence for an edge separating red and brown plastids from green plants within the phylogenetic tree is due to differences in nucleotide composition. The remaining apparent evidence is likely to be due, at least in part, to differences in the distribution of sites free to vary. We discuss the implications of this study for hypotheses of plastid origins. Received: 21 May / 8 September 1998     

The trpA gene on the plastid genome of Cyanidium caldarium strain RK-1

The trpA gene (for the subunit of tryptophan synthase) was found on the plastid genome of the primitive unicellular red alga Cyanidium caldarium strain RK-1. This is the first example of an actively-transcribed gene for tryptophan synthase encoded on a plastid genome. In contrast to trpA, trpB (the gene for the subunit of tryptophan synthase) was encoded in the cell nucleus. Considering the primitive characteristics of C. caldarium, trpB must have been lost from the plastid genome before trpA.The nucleotide sequence data reported in this paper will appear in the DDBJ, EMBL and GenBank Nucleotide Sequence Databases with the following accession number D17791     

The cyanobacterial origin and vertical transmission of the plastid tRNALeu group-I intron

We have surveyed the distribution and reconstructed the phylogeny of the group-I intron that is positioned in the anticodon loop of the tRNA^Leu gene in cyanobacteria and several plastid genomes. Southern-blot and PCR analyses showed that the tRNA^Leu intron is found in all 330 land plants that were examined. The intron was also found, and sequenced, in all but one of nine charophycean algae examined. Conversely, PCR analyses showed that the tRNA^Leu group-I intron is absent from the red, cryptophyte and haptophyte algae, although it is present in three members of the heterokont lineage. Phylogenetic analyses of the intron indicate that it was present in the cyanobacterial ancestor of the three primary plastid lineages, the Rhodophyta, Chlorophyta, and Glaucocystophyta. Its present-day distribution in plastids is consistent with a history of strictly vertical transmission, with no losses in land plants, several losses among green algae, and nearly pervasive loss in the Rhodophyta and its secondary derivatives. Received: 1 August / 22 September 1999     

Structure and expression of cytochrome f in an Oenothera plastome mutant

Summary The chloroplast mutant pm7 is one of a number of mutants derived from the plastome mutator (pm) line of Oenothera hookeri, strain Johansen. Immunoblotting showed that this mutant accumulates a protein that is cross-antigenic with cytochrome f, but five kilodaltons larger than the mature wild-type protein. Since cytochrome f is known to be translated on plastid ribosomes as a precursor with an amino-terminal extension, it is proposed that the unprocessed cytochrome f precursor accumulates in pm7. In addition to this precursor-sized cytochrome f protein, some mature-sized cytochrome f was also found in the mutant plastids. The pm7 mutation is inherited in a non-Mendelian fashion; but no alterations in chloroplast DNA restriction patterns, or differences in DNA sequence in the region encoding cytochrome f, were found in a comparison of the wild-type and pm7 chloroplast DNAs. Although the mutant was capable of synthesizing heme, no covalently-bound heme, normally found associated with mature, functional, cytochrome f was detected in the mutant at sizes expected for the presumed precursor, or for mature cytochrome f. These results indicate that the aberrant accumulation of a precursor-sized cytochrome f in pm7 is not due to a lesion directly in the plastid gene encoding cytochrome f, petA, or to a deficiency in the ability of the mutant plastids to synthesize or accumulate heme.     

Plastid inheritance in Epilobium

Summary Interspecific hybrids of various Epilobium species have been produced in order to analyse plastid inheritance using restriction fragment polymorphisms of plastid DNA as markers. This analysis reveals that interspecific hybrids exhibit only the fragment pattern of the maternal plastome. Southern hybridization experiments using cloned species-specific plastid DNA fragments as markers confirm the maternal type of plastid inheritance in Epilobium, while providing at least a tenfold increase of sensitivity to detect restriction polymorphisms. Within the limit of detection even young seedlings contain no plastid DNA from the paternal parent. However, investigations of plastomes of large populations have provided evidence that a very low frequency of paternal plastid transmission can occur. Thus, the mechanism which ensures the elimination of paternal plastids is not 100% efficient. This suggestion is also supported by intraspecific reciprocal crosses between plants carrying mutant white and normal green plastids. While the offspring usually exhibit the maternal plastid type, a few cases indicate an apparent paternal plastid transmission.Abbreviations kbp kilobase pairs - ptDNA plastid DNA     

Maize BMS cultured cell lines survive with massive plastid gene loss

As part of developing an ex planta model system for the study of maize plastid and mitochondrial gene expression, a series of established Black Mexican Sweet (BMS) suspension cell lines was characterized. Although the initial assumption was that their organelle biochemistry would be similar enough to normal in planta cells to facilitate future work, each of the three lines was found to have plastid DNA (ptDNA) differing from control maize plants, in one case lacking as much as 70% of the genome. The other two BMS lines possessed either near-wild-type ptDNA or displayed an intermediate state of gene loss, suggesting that these clonal lines are rapidly evolving. Gene expression profiles of BMS cells varied dramatically from those in maize leaf chloroplasts, but resembled those of albino plants lacking plastid ribosomes. In spite of lacking most plastid gene expression and apparently mature rRNAs, BMS cells appear to import proteins from the cytoplasm in a normal manner. The regions retained in BMS ptDNAs point to a set of tRNA genes universally preserved among even highly reduced plastid genomes, whereas the other preserved regions may illuminate which plastid genes are truly indispensable for plant cell survival.Communicated by F.-A. Wollman