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 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.     

Molecular analysis of mitochondrial DNA and its inheritance in Epilobium

Summary The mitochondrial genome of four Epilobium species has been characterized by restriction analysis and hybridizations with gene probes from Oenothera. Mitochondrial DNA of Epilobium has a complex restriction fragment pattern and an estimated size of about 320 kb. All species exhibit specific restriction patterns. Plasmid-like DNA molecules of 0.3 kb to 1.2 kb are found in preparations of undigested nucleic acids of mitochondria from E. montanum, E. watsonii, and E. lanceolatum. In contrast, the mitochondria of E. hirsutum contain double-stranded RNAs of 2.7 kb. The location of the genes for cytochrome c oxidase subunits I and III on the mitochondrial DNA seems to be conserved in those species analyzed. However, the genes for subunit II of this complex, and for the alpha subunit of ATPase, are located on different restriction fragments in the mitochondrial genomes of certain species. The location of the COX II gene on different BamHI fragments in E. watsonii and E. lanceolatum has been used for the analysis of mitochondrial inheritance in reciprocal hybrids. Like the plastids, mitochondria are inherited maternally in Epilobium.Abbreviations kb kilobase pairs - mtDNA mitochondrial DNA     

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.     

Evidence for multiple xenogenous origins of plastids: comparison of psbA-genes with a xanthophyte sequence

Summary When only plastidic features are considered, it is difficult to distinguish between monophyletic and polyphyletic xenogenous origins of plastids. We suggest that a direct comparison of nuclear and plastidic sequence-similarity pattern will help to solve this problem. The D1 amino acid sequence of six major groups of photosynthetic eukaryotes and of the two groups of photosynthetic prokaryotes are now available, including the psbA-gene product from Bumilleriopsis filiformis, which is the first molecular sequence reported for a xanthophycean alga. Evidence is provided for an independent and polyphyletic origin of plastids from five out of the six major taxa of photosynthetic eukaryotes. This conclusion is reached by comparing a plastid-based pattern of D1 similarity with a nucleus-based similarity pattern published recently. Furthermore, the availability of D1 sequences from five eukaryotic algae led to a re-evaluation of the taxonomic position of Prochlorothrix.     

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.     

Ultrastructure of the mature pollen of Michelia figo (Lour.) Spreng. (Magnoliaceae)

The structural organization of the Michelia figo mature pollen was investigated. The pollen wall consisted of an outer exine and an inner intine, the former being coated by a thin polysaccharide pellicle. The intine comprised three structurally distinct layers that were equally thick throughout the pollen surface. The generative cell (GC) was closely associated with the vegetative cell (VC) nucleus and its periplasm was found to maintain communication with the sporoderm through a complex plasmalemmic cord. In the freeze-fixed pollen a fluffy coat was detected on the cytoplasmic face of the VC plasmalemma bordering the GC. The plastids were present in only the VC and usually contained abundant small starch grains. In a few pollen grains, however, little or no starch existed and in this case one or more electron dense inclusions appeared in the plastids. Microbodies were found in both the VC and GC. In the VC they presumably have a glyoxysomal function as indicated by the numerous lipid droplets in the cytoplasm and the spatial relationship of microbodies with lipid droplets and/or mitochondria. In the GC the function of the microbodies is unclear once this cell had no abundant lipid reserves and the microbodies did not show any preferential relationship with other organelles. The most conspicuous feature of the VC cytoplasm was the high amount of storage vacuoles which displayed a striking different appearance after one and the other of the fixation techniques used. In contrast to the chemically fixed pollen they were quite polymorphic in the freeze-fixed pollen, and appeared uniformly filled with fibrillar material. Enzymatic digestion with protease has revealed most of this material to be proteinaceous in nature. The existence of phytin reserves is, however, also probable. These protein storage vacuoles closely resemble those in storage tissues of seeds and fruits.     

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     

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.     

Evolutionary relationship of psbA genes from cyanobacteria,cyanelles and plastids

Summary The psbA gene is part of the reaction center of photosystem II in cyanobacteria and the plastids of higher plants. Its primary sequence is highly conserved among all species investigated so far and its sequence shows homologies with the L and M subunits of the reaction center of photosynthetic bacteria. We have analyzed the psbA homolog from a eukaryotic alga, Cyanophora paradoxa, where the gene is encoded on cyanelle DNA. These cyanelles are surrounded by a murein sacculus and resemble cyanobacteria in many other characteristics, although they are genuine organelles that functionally replace plastids. Analysis of the gene revealed a psbA protein identical in length (360 codons) with the cyanobacterial counterpart. The overall sequence identity is, however, more pronounced between cyanelle psbA and the shorter (353 amino acids) psbA product found in higher plants. These data strongly support the postulated bridge position of cyanelles between chloroplasts and free-living cyanobacteria.     

Intron-encoded open reading frame of the GIY-YIG subclass in a plastid gene

Group-I introns, containing open reading frames (ORFs) that code for homing endonucleases, are widely distributed amongst eukaryotic organellar genomes. However, endonucleases of the GIY-YIG subclass have a restricted distribution in mitochondria and bacteriophages, and have never been observed in plastids. We have found the GIY-YIG motif in an intronic ORF within the previously published psbA gene sequence from Chlamydomonas reinhardtii chloroplasts. Based on phylogenetic analysis and an evaluation of amino-acid substitutions, this ORF is not closely related to any of the other GIY-YIG ORFs. These results suggest that GIY-YIG ORFs have a longer evolutionary history than previously assumed.     

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.     

Extraordinary stability of IgE-binding Parietaria pollen allergens in relation to chemically bound flavonoids

It is known that the skin-active and IgE-binding components in Parietaria pollen extracts are not restricted to the predominant protein allergens of M_r 12 000–15 000, but are present as well among the naturally occurring constituents of M_r < 10 000. Indeed, the IgE-binding Parietaria pollen components are quite heterogeneous, ranging from high- to low-molecular mass, whereby the IgE-binding epitopes display an unusual chemical stability. Furthermore, the pollen of Parietaria species demonstrably contain a high proportion of flavonoid pigments. Since these pollen grains cannot be collected entirely free from non-pollen plant parts, the usual allergenic extracts of Parietaria encompass both the polyphenolic substrate molecules and the enzyme polyphenoloxidase as ingredients for the oxidative generation of flavonol-protein conjugates during the extraction process. In the present work this is illustrated by spectroscopic analyses of the free and bound flavonoids in Parietaria pollen extracts, as well as of the peptide fragments produced from the allergenic proteins by enzymatic or chemical hydrolysis. None of these relatively harsh treatments had a significant effect on the IgE-binding properties of the allergenic (sub-)components, even though detectable proteins in isoelectric focusing and immunoblotting were lost. It is proposed that the extraordinary stability of IgE-binding Parietaria components over a wide molecular range may be attributed to chromophoric flavonoid side-chains as (parts of) the corresponding B-cell epitopes.     

Mitochondria and Apoptosis: HQ or High-Security Prison?

Whether we view the mitochondria as the headquarters for the leader of a crack suicide squad or as a prison for the leader of a militant coup, the role of the mitochondria in the apoptotic process is now well established. During apoptosis the integrity of the mitochondria is breeched, the mitochondrial transmembrane potential drops, the electron transport chain is disrupted, and proteins from the mitochondrial intermembrane space (MIS) such as cytochrome c are released into the cytosol, although not necessarily in that order. In the cytosol, cytochrome c forms part of a proteinaceous complex that directly activates caspase-9, one of the apical enzymes responsible for the dismantling of the cell. In this way a mitochondrial factor which is normally locked away from the rest of the cell can directly trigger apoptosis. The need to regulate the release of cytochrome c suggests that the mitochondria may be the decision center for whether a cell lives or dies. Various hypotheses have been formulated to explain how proteins of the MIS are released and how this process is regulated. These include the Bcl-2-regulated opening of a permeability transition pore or an increase in mitochondrial transmembrane potential followed by outer membrane rupture. It remains to be clarified which mitochondria specific events are essential for apoptosis and which are merely consequences of apoptosis.     

Biased organelle transmission in somatic hybrids ofLycopersicon esculentum andSolanum lycopersicoides

Summary Restriction fragment length polymorphisms (RFLPs) were used to determine the transmission of organelle genomes in somatic hybrid plants of tomato and its wild relativeSolanum lycopersicoides. Biased frequencies of organelle combinations were observed in a population of 70 somatic hybrid plants each derived from a separate callus. The plastids in 68 of 70 hybrids examined were fromL. esculentum. One of the remaining hybrids, plant 240, hadS. lycopersicoides plastids and the other, plant 63, had a mixture of parental plastids. Forty-six of the same 70 plants were analyzed for mtDNA and all had that ofS. lycopersicoides including plant 240. One of these hybrids had novel mtDNA fragments which mayhave resulted from recombination or rearrangement. The biased transmission may have resulted from an initial unequal input of organelles, differential replication of organelles, or nucleo-organelle incompatibility.Michigan Agricultural Experiment Station Journal Article No. 12538. Supported by Grant No. I-751-84R from BARD, The United States-Israel Binational Agricultural Research and Development Fund     

Double-stranded RNA molecules in Brassica are inherited biparentally and appear not to be associated with mitochondria

Summary The large RNAs previously detected in mitochondrial preparations of Brassica plants are shown here to be double-stranded molecules that lack homology to the mitochondrial genome and are synthesized in a DNA-independent manner. In crosses between nuclear isogenic lines, the dsRNAs were transmitted through both seed and pollen, whereas the mtDNA, as expected, was transmitted only from the seed or the maternal parent. In addition, the Brassica dsRNAs did not co-purify with mitochondria during Percoll gradient centrifugation. In identical gradients, however, the maize autonomously replicating species S/Ru-a and S/Ru-b co-purified precisely with mitochondria. The results suggest that, unlike the maize RNAs, the Brassica dsRNAs are not physically or genetically associated with mitochondria and, more generally, that the occurrence of dsRNA in mitochondria may be less widespread than recent reports indicate.     

The ultrastructure of the cyst wall of a murine Sarcocystis

Summary Electron microscope studies of the cyst wall of a murine Sarcocystis-like organism of Mus musculus indicate that the sarcocyst wall is a degenerate muscle cell. The degenerate muscle cell contains nuclei, mitochondria, ribosomes, and myofibrils in disarray with the Z-bands thickened. Surrounding the outside of the muscle cell there is a layer of fibrous material, and occasional fibroblasts are seen. The parasites are found within a parasitophorous vacuole in the muscle cell. There are short finger-like projections extending into the vacuole from the muscle cell. The ground substance of the vacuole is fairly homogeneous, but somewhat more electron dense between the finger-like projections.     

Cryptosporidium parvum Cpn60 targets a relict organelle

Chaperonin 60 (Cpn60) is a well-established marker protein for eukaryotic mitochondria and plastids. In order to determine whether the small double-membrane-bounded organelle posterior to the nucleus in the apicomplexan Cryptosporidium parvum is a mitochondrion, the Cpn60 gene of C. parvum sporozoites (CpCpn60) was analyzed and antibodies were generated for localization of the peptide. Sequence and phylogenetic analyses indicated that CpCpn60 is a mitochondrial isotype and that antibodies against it localize to the rough endoplasmic reticulum-enveloped remnant organelle of C. parvum sporozoites. These data show this organelle is of mitochondrial origin.Communicated by M. Brunner