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.     

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 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     

Plastid inheritance in Pisum sativum L.

Summary Cultivar variability for levels of plastid DNA (cpDNA) in the germ cell line of germinated pea pollen has suggested the possibility of biparental plastid transmission. In order to examine this possibility further, RFLP markers were used to follow the transmission of cpDNA from parents to their F₁ offspring. Results from these inheritance studies clearly indicate the presence of only maternal plastid markers in the F₁ progeny of each cross examined, irrespective of the pollen cpDNA levels of the paternal parent. The same result is obtained for F₁ progeny produced from crosses using pollen characterized by comparatively high cpDNA content, even when offspring are sampled at early developmental stages. Thus, there appears to be little correspondence between pollen cytological data indicating potential paternal plastid transmission and data from molecular marker studies confirming that P. sativum generally follows a uni-parental-maternal mode of plastid inheritance. Insufficient F₁ progeny were examined to exclude instances of trace biparentalism.     

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     

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.     

Paternal plastid inheritance in alfalfa: plastid nucleoid number within generative cells correlates poorly with plastid number and male plastid transmission trength

Summary A previous study on alfalfa determined that the number of plastids/generative cell does not necessarily correlate with male plastid transmission strength in a given genotype. The objectives of the present study were to learn (1) whether plastid nucleoid number/generative cell is comparable to the number of plastids/generative cell, and (2) whether plastid nucleoid number/generative cell correlates with known male plastid transmission behavior in three alfalfa genotypes. Our results, which were based upon 150 generative cells examined by DAPI/epifluorescence microscopy, indicate that the mean plastid nucleoid number/generative cell is much less than the mean number of plastids/generative cell in genotype 7W (60 nucleoids/264 plastids) and genotype 301 (54 nucleoids/165 plastids). In genotype MS-5, mean plastid nucleoid number/generative cell (45) is similar to the mean number of plastids/generative cell (65). The significantly fewer plastid nucleoids/generative cell in MS-5, compared to that of 7W and 301, correlates positively with the relatively poor male plastid transmission strength of this genotype. However, the difference between the mean number of plastid nucleoids/generative cell in 7W and 301 is not significant, yet 301 is a much stronger transmitter of male plastids than is 7W.     

From chloroplasts to “cryptic” plastids: evolution of plastid genomes in parasitic plants

To date, more than 130 plastid genomes (plastomes) have been completely sequenced. Of those, 12 are strongly reduced plastid genomes from heterotrophic plants or plant-related species that exhibit a parasitic lifestyle. Half of these species are land plants while the other half consists of unicellular species that have evolved from photosynthetic algae. Due to their specialized lifestyle, parasitic lineages experienced a loss of evolutionary pressure on the plastid genome and, in particular, on the photosynthesis-related genes. This made them tolerant for the accumulation of detrimental mutations and deletions in plastid genes. That parasitic plants are naturally occurring plastome mutants makes them a rich source of information concerning plastome evolution and the mechanisms that are involved. This review reports on the progress made in recent years with parasitic plant plastomes and attempts to summarize what we can learn from analysing the genomes of functionally reduced, or cryptic, plastids. Particularly, the loss of genes for a plastid-encoded RNA polymerase as well as an intron maturase and the retention of the gene for the large subunit of the Calvin cycle enzyme Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) in selected species will be discussed.     

Evidence for tissue-specific cytosine-methylation of plastid DNA from Zea mays

Summary Total DNA isolated from leaves, etiolated seedlings, roots, endosperm or embryos of Zea mays was digested separately with each of the restriction enzymes HpaII, MspI and HhaI, and the resulting fragment patterns, which were specific for the plastid rRNA operon, were analyzed by Southern hybridization. While most of the fragment patterns were consistent with previously established physical maps, the partial resistance shown by one HpaII site and one HhaI site, both of which reside in the 16S/23S rDNA spacer region, was observed in DNA isolated from embryo, root tissue and endosperm. The partially resistant HpaII site was susceptible to cleavage with restriction enzyme MspI. From this and from the known inhibition of restriction enzyme Hhal at methylated HhaI sites, we conclude that the partial resistance of the two sites is caused by C-specific methylation of plastid DNA in the respective tissues. The tissue specificity of this DNA methylation is likely to reflect a differential expression of plastome encoded genes.     

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     

A circular 73 kb DNA from the colourless flagellate Astasia longa that resembles the chloroplast DNA of Euglena: restriction and gene map

Summary A 73 kbp circular DNA was isolated from the colourless euglenoid flagellate Astasia longa. Restriction sites of 12 restriction endonucleases were mapped on this DNA. Southern hybridization using plastid gene probes from Euglena, spinach and tobacco revealed sequence homologies to the genes coding for 16S and 23S ribosomal RNAs, elongation factor Tu (tufA) and the large subunit of ribulose-1,5-bisphosphate carboxylase (rbcL). The locations of these sequences on the restriction map were determined. Sequences homologous to chloroplast genes psaA, psbA, psbD, psbE and atpA are not present. The ribosomal RNA genes are organized in three tandem repeats, each containing one 23S and one 16S rRNA gene. In addition, there is one extra 16S rRNA gene. These results indicate the presence of a truncated form of a plastid DNA in Astasia.     

Restriction map and clone bank of tomato plastid DNA

Summary Tomato plastid DNA has been isolated from leaf chloroplasts andPst1 fragments cloned in the plasmid vector pUC8. Recombinant plasmids containing all but the largestPst1 fragment were identified by colony hybridisation. A restriction map of the plastid DNA for four restriction endonucleases was generated by digestion of the clonedPst1 fragments and by hybridisation to Southern blots. The plastid DNA is a circular molecule of 156.6–159.4 kbp with a large inverted repeat, and shows high conservation of restriction sites with plastid DNA from tobacco andPetunia.     

DNA markers define plastid haplotypes in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

To identify genetic markers in the Arabidopsis thaliana plastid genome (ptDNA), we amplified and sequenced the rpl2-psbA and rbcL-accD regions in 26 ecotypes. The two regions contained eight polymorphic sites including five insertions and/or deletions (indels) involving changes in the length of A or T mononucleotide repeats and three base substitutions. The 27 alleles defined 15 plastid haplotypes, providing a practical set of ptDNA markers for the Columbia, Landsberg erecta and Wassilewskija ecotypes that are commonly used in genetic studies and also for the C24 and RLD ecotypes that are the most amenable for cell culture manipulations.     

Physical mapping of the plastid genome from the chlorophyll c-containing alga,Cryptomonas Φ

Summary A physical map of the circular plastid genome of Cryptomonas has been constructed using the enzymes SacI, BamHI, SmaI, SalI, PstI and XhoI. In addition, fine-structural mapping of the inverted repeat region has been performed using AvaI, BglII, EcoRI and XbaI. The inverted repeat is very small, encompassing no more than 6 kb and containing only genes for the rRNAs. It divides the plastid genome into a small singlecopy region of 12–13 kb which contains genes for phycoerythrin and the 32 kd photosystem II polypeptide, and a large single-copy region of 93–94 kb, giving a total size of 118 kb. The genes for the large subunit of ribulose-1,5-bisphosphate carboxylase (Rubisco) and the beta subunit of ATP synthase CF₁ are encoded in the large single-copy region. The evolutionary significance of the organization of this plastid genome, the first presented from the chlorophyll c-, phycobiliprotein-containing group of algae, is discussed.     

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     

Plastid DNA from Pyrenomonas salina (Cryptophyceae): physical map,genes, and evolutionary implications

Summary Cryptomonads are thought to have arisen from a symbiotic association between a eukaryotic flagellated host and a eukaryotic algal symbiont, presumably related to red algae. As organellar DNAs have proven to be useful tools in elucidating phylogenetic relationships, the plastid (pt) DNA of the cryptomonad alga Pyrenomonas salina has been characterized in some detail. A restriction map of the circular 127 kb ptDNA from Pyrenomonas salina was established. An inverted repeat (IR) region of about 5 kb separates two single-copy regions of 15 and 102 kb, respectively. It contains the genes for the small and large subunit of rRNA. Ten protein genes, coding for the large subunit of ribulose-1,5-bisphosphate carboxylase, the 47 kDa, 43 kDa and 32 kDa proteins of photosystem II, the ribosomal proteins L2, S7 and S11, the elongation factor Tu, as well as the - and -subunits of ATP synthase, have been localized on the restriction map either by hybridization of heterologous gene probes or by sequence homologies. The gene for the plastidal small subunit (SSUr) RNA has been sequenced and compared to homologous SSU regions from the cyanobacterium Anacystis nidulans and plastids from rhodophytes, chromophytes, euglenoids, chlorophytes, and land plants. A phylogenetic tree constructed with the neighborliness method and indicating a relationship of cryptomonad plastids with those of red algae is presented.     

Restriction and gene maps of plastid DNA from Capsicum annuum

Summary Chloroplasts and chromoplasts were isolated from green and red fruits, respectively, of the bell pepper, Capsicum annuum var. Emerald giant. A comparison of the restriction patterns of DNAs isolated from these plastids was made using single and double digests by SacI, PvuII, PstI, and SalI and found to be indistinguishable. It is inferred therefore that the conversion of chloroplasts to chromoplasts in Capsicum annuum does not involve any large rearrangements of the plastid chromosome. A restriction map of Capsicum annuum plastid DNA was constructed using the same restriction enzymes in single digests and in all possible pair combinations. Overlapping restriction fragments were identified by digesting each product of a single digest with each of the other three enzymes. The resulting restriction map is similar to that of chloroplast DNA from other members of the Solanaceae with respect to most restriction sites. The genome size corresponds to 143 kbp. The locations of 24 genes, coding for ribosomal RNAs and for proteins of Photosystem I (PSI), Photosystem II (PSII), ATP synthase, cytochromes, the large subunit of ribulose-1,5-bisphosphate carboxylase-oxygenase (E.C. 4.1.1.29) (RuBPC), and ribosomal proteins were determined by probing Southern blots of Capsicum chloroplast DNA with probes of genes from spinach and tobacco. The gene locations are completely conserved with respect to those of other members of the Solanaceae and the majority of higher land plants.Abbreviations and notations atpA, atpB, atpE, and atpF genes for the , , , and I subunit, respectively, of ATP synthase - cpDNA chloroplast DNA - petA, petB, petD genes for cytochrome f, cytochrome b ₆, and subunit IV of cytochrome b ₆/f complex, respectively - psaA, psaB, psaC genes for the P₇₀₀ apoproteins - psbA gene for Q_B - psbB and psbC genes for the 51-kDa and 44-kDa proteins, respectively, of PSII - psbD gene for the Q_B-like polypeptide of PSII - psbE gene for cytochrome b ₅₅₉ - rbcL gene for the large subunit of RuBPC - rpl2 gene for ribosomal protein L2 - rpoA gene for the subunit of RNA polymerase - rps11, rps12 and rps19 genes for ribosomal proteins S11, S12, and S19, respectively - rps19 open reading frame for a protein with N terminus similar to that of S19 - RuBPC ribulose-1,5-bisphosphate carboxylase-oxygenase (E.C. 4.1.1.29) - trnH gene for histidine transfer RNA - URF39 and URF509 unidentified reading frames for polypeptides of 39 and 509 amino acids, respectively Gene names follow the convention of Hallick and Bottomley (1983)     

Loss of transfer RNA genes from the plastid 16S–23S ribosomal RNA gene spacer in a parasitic plant

Summary The plastid 16S–23S intergenic spacer region in Conopholis americana, a totally heterotrophic angiosperm in the family Orobanchaceae, has undergone large deletions, including the entire tRNA^Ile gene and all but small remnants of the tRNA^Ala gene. The length of the region is less than 20% of that of other land plants which have been investigated, making it the smallest 16S–23S intergenic spacer reported thus far for any land plant. The remaining sequences in the spacer are 90.1% identical to tobacco, indicating that, while the region is well conserved at the sequence level, it is evolving rapidly by deletion. Experiments using the polymerase chain reaction and hybridization to DNA gel blots have failed to revcal either of the two missing tRNA genes elsewhere in the Conopholis cell.     

Lack of a functional plastid tRNACys gene is associated with loss of photosynthesis in a lineage of parasitic plants

Summary We recently reported that the gene for chloroplast tRNA^Cys(GCA) is a pseudogene in the plastid DNA of Epifagus virginiana, a non-photosynthetic parastic flowering plant in the family Orobanchaceae. Since this is the only tRNA^Cys gene in the plastid genome, and since Epifagus appears to possess a functional plastid translational apparatus, it seems probable that nuclear-encoded tRNAs are imported into plastids to effect translation. In this study we have surveyed species closely related to Epifagus to establish how widespread the loss of this tRNA gene has been. We find that Conopholis americana, another non-photosynthetic parasite, lacks the gene altogether, but that seven closely-related photosythetic plants (both parasitic and free-living) maintain an intact chloroplast tRNA^Cys gene. Thus, the tRNA^Cys gene appears to have become non-functional at the same time that photosynthetic ability was lost. This may be because the levels of putatively imported tRNAs are sufficient to meet the demands of plastid gene expression under nonphotosynthetic conditions only.