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Structural and physiological studies of a plastome cotton mutant with slow sorting out
Abstract
Structural and physiological investigations of a variegated plastome mutant of cotton ( Gossyplum hirsutum L.) first found in 1964 were undertaken to determine the nature of the lesion in the mutant and possible reasons for the relatively long persistence of the variegation. Mutant plastids were much less structurally complex even at early developmental stages and large differences in chloroplast ribosome density were noted. Only single, or slightly doubled thylakoids were noted in the mature mutant plastid. At later stages the pigments from the mutant tissue were bleached and the thylakoid swelled to form small “plastid vacuoles.” Numerous mixed cells, single cells with both mutant and clearly wild-type plastids, were present in much of the mosaic tissue. However, unlike other plastome mutants with persistent mixed cells, no fusions between wild-type and mutant plastids were noted. We suggest that the persistence of mixed cells in this cotton mutant is due to the sorting out of both chloroplast DNA molecules as well as the two chloroplast types. Electrophoretic studies of chloroplast proteins indicate that many chloroplast-encoded and nuclearly encoded polypeptides are present relatively less in the mutant but polypeptides in the Mr range from 55–100 kD are present in increased amounts compared to the green chloroplasts. Although some cyclic electron flow through cytochrome f was noted in the mutant tissue, the fluorescence transients and the lack of grana stacks indicate a lack of photosystem II activity.
... Il est important de noter que seul le premier niveau -etéventuellement le deuxième niveau en cas d'évènement de fusion entre organites -peut donner lieuà une recombinaison entre différents génomes. L'hétéroplasmie peut exister simultanément a plusieurs niveaux, comme cela aété montré chez un mutant de coton panaché comportant des cellules aux plastes normaux, des cellules aux plastes blancs, et des cellules avec un mélange de plastes (Lax et al., 1987). ...
... Au contraire, chez Senecio vulgaris, Frey (1999) a observé que l'hétéroplasmieétait très bien maintenue d'une générationà l'autre, même lorsque les plantes n'étaient pas traitéesà la triazine (ce maintien est etonnant car la mutation résistante est associéeà un fort coût et devrait doncêtre rapidement contre-sélectionnée). Un cas de transmission intermédiaire aégalement eté observé chez un mutant panaché du coton. Lax et al. (1987) ont montré que l'hétéroplasmieétait transmise de mère en descendants mais qu'ilétait peu probable que l'hétéroplasmie puisseêtre maintenue pendant de nombreuses générations, les variations inter-générationnelles dans la proportion des deux types de chloroplasté etant trop importantes. Par ailleurs, le rôle de la reproduction sexuée dans la ségrégation de l'hétéroplasmie aété démontré dans deuxétudes, l'une sur l'hétéroplasmie mitochondriale chez l'olivier (Olea europaea) et l'autre sur l'hétéroplasmie chloroplastique chez le kiwi (Actinia deliciosa). ...
... In plants, simple mathematical models showed that, when heteroplasmy occurs, vegetative sorting of organelles should be complete within one plant generation (Birky 2001). But experimental studies are rare and results are controversial : Some showed a complete sorting in one generation that occurred during development (Erickson and Kemble 1993 ;Matsushima et al. 2008) or from a bottleneck in the germ line (Chat et al. 2002) but heteroplasmy could also be maintained, by drift or selection, and transmitted to offspring (Lax et al. 1987 ;Frey 1999). In Silene vulgaris, heteroplasmic mothers produced on average 17% of heteroplasmic offspring, showing that vegetative sorting occurred but was not always complete in one generation (Pearl et al. 2009 ;Bentley et al. 2010). ...
In flowering plants, gynodioecy -- a system in which females and hermaphrodites coexist within populations -- is the most common sexual system after hermaphroditism. The evolution and maintenance of gynodiocy and its underlying polymorphism have puzzled evolutionary biologists since the 19th century. The main principles of its evolution are well known but some points remain vague. During my PhD, I explored three aspects of gynodioecy using an experimental approach in the species Silene nutans and a theoretical approach. First, I studied the genetic determination of gynodioecy using controlled crosses that showed that the genetic determination of sex was cytonuclear, i.e. controlled by several cytoplasmic male sterility (CMS) genes and several nuclear restorers of fertility. Second, I focused on gynomonoecious plants -- those that carry both pistillate (female) flowers and perfect (hermaphrodite) flowers -- that are frequently found in Silene nutans as in other gynodioecious species. I showed that the floral and reproductive traits of this third sex phenotype were often intermediate between those of females and hermaphrodites but varied with varying proportions of pistillate flowers on the plant. Contrary to what was previously thought, the plasticity of the gynomoneocious phenotype was found to be limited, suggesting a genetic determination whose characterization is still in progress. The third part of my PhD was motivated by recent evidences of heteroplasmy -- the coexistence of different mitochondrial genomes within an individual -- and occasional transmission of the mitochondrial genome through pollen in Silene vulgaris. I showed theoretically that the occurrence of a cytoplasmic male sterility gene can favor the evolution of paternal leakage of mitochondria. I also investigated mitochondrial inheritance in Silene nutans by genotyping progenies from controlled crosses.
... Heteroplasmy has been reported in mitochondrial genomes and cp-genomes in many organisms such as human, animals and plants (as reviewed in [30]). Chloroplast heteroplasmy is believed to originate from bi-parental inheritance, a slow rate of spontaneous mutation or recombination of the cp-genome, or incomplete cpgenome sorting which happen in the cp-genome [30][31][32][33], although the genome itself is considered to be highly conservative. Despite the fact that there are thousands of cp-genome copies in the cells, heteroplasmy was once thought to be uncommon in higher plant cpgenomes, instead the homogeneity of these copies, known as homoplasmy, was more often observed and assumed [29,30]. ...
... N.V. Hoang et al. / New Negatives in Plant Science 1-2 (2015)[33][34][35][36][37][38][39][40][41][42][43][44][45] ...
Background
The chloroplast genome of plants has been frequently sequenced using chloroplast DNA derived by techniques involving chloroplast isolation and or by PCR amplification using primer sequences targeted to amplify the chloroplast genome. Using these approaches, chloroplast heteroplasmy, described as variations in the chloroplast sequence within an individual plant, has been reported in many plant species. More recently, next generation sequencing (NGS) technologies have allowed chloroplast genome sequences to be extracted from shotgun sequences of total plant DNA.
Results
Here, we used DNA preparations varying in nuclear, mitochondrial and chloroplast enrichment to explore the potential to distinguish genuine chloroplast heteroplasmy from apparent heteroplasmy due to sequence-variant homologues of chloroplast genome sequences inserted in nuclear or mitochondrial genomes. Application of NGS to the whole sugarcane genome followed by read mapping analysis of the complex sugarcane system allowed the assembly of a complete chloroplast genome sequence of sugarcane cv. Q155. Variant analysis showed that they were present only at frequencies that could be attributed to homologues of chloroplast sequences inserted in the nucleus or mitochondria.
Conclusions
The result suggests that earlier reports of heteroplasmy in chloroplasts may have been due to contaminating sequences from other genomes (nuclear or mitochondrial) in chloroplast preparations or specific amplification of sequences from these genomes. This demonstrates that the ability to evaluate sequence abundance avoids the risks of attributing a chloroplast gene homologue from the nucleus or mitochondria to the chloroplast.
... Many of the cases of variegation known to date in cultivated plants are due to spontaneous mutations of the plastid genome ( Kirk and Tilney-Bassett 1978). But heteroplasmy can also arise from biparental inheritance when each parent transmits its organelles to the zygote, as has been observed, for example, in Pelargonium ( Tilney-Bassett and Birky 1981) or from uniparental inheritance when sorting-out in the parent is incomplete so that some heteroplasmic gametes are produced, as observed in Gossypium ( Lax et al. 1987) and in Epilobium (Michaelis 1962). So far the extent of heteroplasmy has been difficult to estimate, as its detection was limited to the availability of phenotypic markers. ...
... Even if rare, the possibility of germ cells being heteroplasmic cannot be completely excluded. In plants, a plastid heteroplasmy was found to persist after several generations of sexual propagation in Gossypium ( Lax et al. 1987) and in Senecio ( Frey 1999). Hypothesizing heteroplasmy of the germ line cells of D uno, one possible explanation for the fixation of one cpDNA within a single sexual generation consists of drastic drift following a ''germ-line bottleneck'' (Chinnery et al. 2000). ...
... For example, Moon et al. [16] claimed the existence of a persistent cpDNA heterogeneity in single rice plants. Lax et al. [10] have described a plastome mutant of cotton that exhibits a slow rate of sorting out, resulting in a persistence of mixed cells possessing both wild type and mutant plastids. Heteroplasmy is possible in several species of conifers that exhibit paternal plastid inheritance [33, 34] . ...
... In rice, single plants were reported by Moon et al. [16] to maintain a relatively constant rate of two cpDNA types, although evidence for both forms of the putative rice cpDNA being of true chloroplast origin is not entirely convinc- ing [17]. Lax et al. [10] studied sorting out in variegated cotton containing a plastome mutant, and suggested that the high frequency of plants heterozygous for cpDNA was the result of a slow sorting out, possibly facilitated by plastid fusions that produced within-plastid DNA heterogeneity. We could not measure sorting out rate, both because our cpDNA mutants did not result in chlorophylldeficient plants and because it is unlikely that all plants assayed were even fertilized by pollen possessing a different cpDNA type. ...
Two chloroplast DNA (cpDNA) regions exhibiting a high frequency of intra- or inter-species variation were identified in 12 accessions of the genus Medicago. Restriction maps of both regions were prepared for alfalfa, and the probable nature of the events causing the DNA differences was identified. Specific DNA fragments were then cloned for use in identification of variants in each region. Two each of M. sativa ssp. varia and ssp. caerulea and one of six M. sativa ssp. sativa single plants examined possessed cpDNA heterogeneity as identified by screening extracts for fragments generated by the presence and absence of a specific Xba I restriction site. Three plants of M. sativa ssp. sativa, two of each of sspp. varia and caerulea, and three M. scutellata were also examined for single-plant cpDNA heterogeneity at a hypervariable region where differences resulted from small insertion-deletion events. A single M. scutellata plant with mixed cpDNAs was identified. Sorting out was seen when one spp. sativa plant with mixed plastid types identifiable by the Xba I restriction site difference was vegetatively propagated. This indicated that the initial stock plant was heteroplastidic. Controlled crosses will be required in order to test whether heteroplasmy results from chloroplast transmission in the pollen and to examine the dynamic of sorting out. However, heteroplasmy is apparently not a rare situation in Medicago.
... microsatellites chloroplastiques) qui pourraient être à l'origine d'un polymorphisme intraplante . De même, l'état hétéroplasmique est réputé transitoire, mais une hétéroplasmie stable par voie sexuée et asexuée a déjà été signalée chez les végétaux (Lax et al. 1987;Frey 1999). Malgré cela, l'hérédité biparentale reste fréquemment invoquée pour expliquer l'émergence de cas d'hétéroplasmie plastidique dans le règne végétal et c'est une des raisons pour laquelle nous nous y sommes intéressés dans le cadre de la thèse. ...
*INRA CR Bordeaux, Documentation de Centre Diffusion du document : INRA CR Bordeaux, Documentation de Centre Diplôme : Dr. Ing.
... Heteroplasmy was present in the chloroplast genomes of many C. roseus varieties in the present study (Supplemental Table S1). The chloroplast heteroplasmy is a phenomenon that has been detected in many flowering plants, for example, Pelargonium [43], Gossypium [44], Oenothera [45], Medicago [46], Actinidia [47], Cynomorium [48], Passiflora [49] and Phoenix dactylifera [16]. However, a concern has been raised about the commonness of this phenomenon in chloroplast genomes. ...
Catharanthus roseus, an important medicinal plant, is known for the production of various pharmaceutical compounds including the anti-tumour drugs, vinblastine, and vincristine. Also, this ornamental plant is widely known for its flowers with different colours. Its varieties are identified on the basis of the morphological characteristics of the flowers, including petals and flower eye colour. Morphological characterization cannot be performed before the flowering stage, leading to a major obstacle for consumers, since most of the sales associated with its medicinal value occur at the vegetative stage. In the present study, we utilized high-throughput, next-generation sequencing to detect chloroplast single nucleotide polymorphism (SNP) that are unique to each variety for molecular characterization. The total genomic DNA of eight C. roseus varieties were sequenced using Illumina HiSeq 2000 platform. The alignment of resulting sequences to chloroplast reference genome showed the presence of specific SNPs for all eight varieties. Also, intravarietal SNPs were found that confirmed the applicability of heteroplasmy theory in the chloroplast genome of this species. Thus, this investigation provides valuable insights into molecular characterization of C. roseus, especially at the vegetative stage.
... Chloroplast heteroplasmy is believed to originate from bi-parental inheritance, a slow rate of spontaneous mutation or recombination of the cp-genome, or incomplete cp-genome sorting, which happens in the cp-genome ( Lax et al., 1987, Corriveau and Coleman, 1988, Chat et al., 2002, Frey et al., 2005), although the genome itself is considered to be highly conservative. Despite the fact that there are thousands of cp-genome copies in the cells, heteroplasmy was once thought to be uncommon in higher plant cp-genomes. ...
Sugarcane (Saccharum spp. hybrids) is a leading industrial crop in tropical and subtropical regions worldwide. More recently, sugarcane has been selected as a key feedstock for biofuels due to its rapid growth, high fiber content and favorable energy input/output ratio. Breeding sugarcane varieties with biomass for efficient conversion to biofuels can be optimized by understanding the genetic control of biomass composition. However, the genetic analysis of these traits is hindered by the genomic complexity, and the limited availability of a reference genome. The aims of this project were: the development of a high-throughput profiling method for rapid screening of the key biomass traits in a sugarcane population; the construction of a new full-length transcriptome reference database; and the identification of transcripts associated with sugar and fiber accumulation in sugarcane.
For the screening of genotypes, newly developed predictive models employing near-infrared (NIR) spectral analysis, coupled with the high performance liquid chromatography (HPLC), were shown to allow high-throughput profiling of major components in the fiber and sugar fractions in sugarcane biomass. Contrasting genotypes of low fiber and high fiber (minimum of ~29% and maximum of 61% total dry biomass) were identified amongst 331 samples from 186 sugarcane genotypes. The population studied exhibited a wide range of fiber/sugar ratio, from 0.4 (as low as that of the typical commercial sugarcane variety) to 2.2 (similar to that of energy-cane). In addition, the lignin content (the central factor in the biomass recalcitrance) ranged from 6 to 14% of the total dry biomass. To aid genotyping, a new sugarcane transcriptome (termed as SUGIT database) was constructed using PacBio full-length isoform sequencing (Iso-Seq), and a cDNA library derived from 22 diverse sugarcane genotypes, of the key tissues (leaf, internode and root), at different developmental stages (from immature to mature). Comparative analysis showed that this new SUGIT database included more full-length transcripts, longer predicted transcripts, and higher average length of the largest 1,000 proteins, compared to a de novo assembly from Illumina RNA-Seq short-read data from the same sample set. The annotation suggested that the majority (~94%) of the SUGIT database was from coding RNAs, while a very small proportion (~2%) could be long non-coding RNAs. About 70-82% of the RNA-Seq reads from different tissues mapped back to the SUGIT database, suggesting that it represented well the targeted tissues, while about 69% of this database was aligned with the sorghum genome, confirming the high conservation of orthologs in the genic regions of
ii
the two genomes. Applying the SUGIT database to differential expression analysis (FDR, false discovery rate corrected p-value <0.05), 1,649 transcript isoforms were identified as being differentially expressed between the young and mature tissues in the sugarcane plant, while 555 transcript isoforms were differentially expressed between the high and low fiber genotype groups. The differentially expressed transcripts included those involved in the carbon partitioning between the cell-wall components and sugars, cell function, hormone metabolism, transcription factors, disease/stress resistance, and development.
Taken together, the new NIR- and HPLC-based method evaluated in this thesis allowed the rapid profiling of a large number of sugarcane biomass samples. The SUGIT database facilitated the analysis of differential gene expression at the transcriptional level, defined different full-length isoforms, and predicted transcripts that could be used to improve the sugarcane gene models. Finally, the study identified the candidate transcript isoforms that regulate the accumulation of biomass major components (sugars and fiber) at transcriptional level in sugarcane.
... The traditional view has been that heteroplasmy in plastids is uncommon [25] but several examples of this phenomenon have been detected across flowering plants, including in Actinidia [26], Coreopsis [34], Cynomorium [35], Epilobium [36], Medicago [37], [38], Gossypium [39], Oenothera [40] Oryza [41], Passiflora [42], Pelargonium [43], and Senecio [27]. Thus, heteroplasmy is more common than previously thought and it likely went undetected because of the paucity of molecular studies that examined intra-individual variation. ...
Date palm is a very important crop in western Asia and northern Africa, and it is the oldest domesticated fruit tree with archaeological records dating back 5000 years. The huge economic value of this crop has generated considerable interest in breeding programs to enhance production of dates. One of the major limitations of these efforts is the uncertainty regarding the number of date palm cultivars, which are currently based on fruit shape, size, color, and taste. Whole mitochondrial and plastid genome sequences were utilized to examine single nucleotide polymorphisms (SNPs) of date palms to evaluate the efficacy of this approach for molecular characterization of cultivars. Mitochondrial and plastid genomes of nine Saudi Arabian cultivars were sequenced. For each species about 60 million 100 bp paired-end reads were generated from total genomic DNA using the Illumina HiSeq 2000 platform. For each cultivar, sequences were aligned separately to the published date palm plastid and mitochondrial reference genomes, and SNPs were identified. The results identified cultivar-specific SNPs for eight of the nine cultivars. Two previous SNP analyses of mitochondrial and plastid genomes identified substantial intra-cultivar ( = intra-varietal) polymorphisms in organellar genomes but these studies did not properly take into account the fact that nearly half of the plastid genome has been integrated into the mitochondrial genome. Filtering all sequencing reads that mapped to both organellar genomes nearly eliminated mitochondrial heteroplasmy but all plastid SNPs remained heteroplasmic. This investigation provides valuable insights into how to deal with interorganellar DNA transfer in performing SNP analyses from total genomic DNA. The results confirm recent suggestions that plastid heteroplasmy is much more common than previously thought. Finally, low levels of sequence variation in plastid and mitochondrial genomes argue for using nuclear SNPs for molecular characterization of date palm cultivars.
... It can decrease the utility or even declassify some types of markers for barcoding. There are three mechanisms leading to heteroplasmy: spontaneous mutations of the plant genome (Tilney-Bassett 1978), biparental inheritance (Metzlaff et al. 1981) or uniparental inheritance, but with incomplete sorting-out, for example, during hybridization processes (Lax et al. 1987). In this respect, maternal inheritance had been already confirmed for the genus Cistus . ...
The genus Cistus is a widespread family growing around the Mediterranean area. There is a unique natural population of C. heterophyllus subsp. carthaginensis in Europe (Murcia, Spain) containing 22 individuals. Morphology of these plants suggests the co-occurrence of two distinct types within the population. One type would resemble C. heterophyllus, and a second type would be the result of hybridization events between this endangered population and the locally abundant Cistus albidus. These hybrids have been described in Africa as C. × clausonis. We have analyzed sequences of the chloroplast genes trnK-matK, rbcL, rpoB, rpoC1 and two intergenic regions, trnL-F and trnH-psbA. Surprisingly, we observed heteroplasmy for rpoB and rpoC1 genes in C. heterophyllus and the local C. × clausonis, but not in C. albidus or C. monspeliensis. We found two distinct alleles of rpoB, one present in all species and a second present only in C. heterophyllus and the local C. × clausonis. We also detected two alleles of rpoC1, one common to all species analyzed and a second present only in the local C. × clausonis. Our results show that there is a distinctive rpoB allele common to C. heterophyllus and C. × clausonis from Africa and Europe. The unique rpoC1 allele found in the local C. × clausonis indicates a different origin of this small population, indicating it is not a hybrid formed with the C. albidus or C. heterophyllus currently present in this location.
... Parker' reacted sequentially with anti-RuBisCo, goat anti-rabbit labeled with peroxidase, and a peroxidase incubation media resulted in strong immunostaining of the large subunit of RuBisCo (Fig. 7). Like other anti-RuBisCo sera prepared to the holoenzyme (3), this anti-tobacco RuBisCo (5) recognizes only the large subunit. Although Zemel and Gepstein (17) found that protease inhibitors were required to preserve guard cell RuBisCo, the relatively simple homogenization media used in this report preserved an apparently unaltered large subunit of RuBisCo. ...
Two immunological approaches were used to determine if ribulose bisphosphate carboxylase oxygenase (RuBisCo) is present in guard cell chloroplasts. Immunocytochemistry on thin plastic sections using tissue samples that were processed using traditional glutaraldehyde/osmium fixation and then restored to antigenicity with metaperiodate treatment, resulted in labeling over wild-type mesophyll and guard cell plastids of several green and white variegated Pelargonium chimeras. The density of immunogold labeling in guard cell chloroplasts was only about one-seventh of that noted in mesophyll chloroplasts on a square micron basis. Because guard cell chloroplasts are much smaller than mesophyll chloroplasts, and occur at lower quantities/cell, the relative differences in RuBisCo concentration between the cell types indicate that guard cells have only 0.48% of the RuBisCo of mesophyll cells. No reaction was noted over 70S ribosomeless plastids of these chimeras even though adjacent green chloroplasts were heavily stained, indicating the high specificity of the reaction for RuBisCo. Spurr's resin gave the most successful colloidal gold labeling in terms of low background staining and structural detail but L. R. White's resin appeared to be superior for antigen retention. In the white leaf edges of the white and green Pelargonium chimeras, the only green, functional chloroplasts are in the guard cells. When either whole tissue or plastid enriched extracts from this white tissue were electrophoresed, blotted, and probed with anti-RuBisCo a large subunit band was detected, identical to that in the green tissue. These data indicate that a low, but detectable, level of RuBisCo is present in guard cell chloroplasts.
... Even if rare, the possibility of germ cells being heteroplasmic cannot be completely excluded. In plants, a plastid heteroplasmy was found to persist after several generations of sexual propagation in Gossypium ( Lax et al. 1987) and in Senecio ( Frey 1999). Hypothesizing heteroplasmy of the germ line cells of D uno, one possible explanation for the fixation of one cpDNA within a single sexual generation consists of drastic drift following a ''germ-line bottleneck'' (Chinnery et al. 2000). ...
We report the first case of plastid chimera within the Actinidia genus, where plastid inheritance was believed to be paternal. The heterogeneity of chloroplast DNA observed in the hexaploid Actinidia deliciosa cultivar D uno involves the presence or absence of a particular MspI restriction site in the region between the psbC gene and the tRNA-Ser(UGA) gene. The heterogeneity was first observed using restriction fragment length polymorphism and then confirmed through cloning and sequencing. The analysis of the cloned fragments revealed the presence of two haplotypes: the most frequent type was found in 123 (88.5%) out of a total of 139 colonies screened. Partial sequences of the psbC-trnS fragment from both haplotypes revealed that the polymorphism occurs within the coding region of the psbC gene and consists of a synonymous transition. A contamination-free cross involving D uno as the male parent produced only plants characterized by the most frequent haplotype, indicating either selection bias against the rare type or more likely fixation of the frequent type in tissues leading to the formation of the male gametes. The MspI restriction profiles performed on various tissues suggest that the rarer type is absent from the histogenic layer LII and that D uno is a periclinal plastid chimera.
... The fact that plastids rarely fuse (Sears 1980) must limit the effective number of plastid genomes per cell. The observation that rare cases of plastid fusion seem to account for unusually slow sorting out (Vaughn 1981; Lax et al. 1987 ) supports this view. Transient tubular connections between chloroplasts within cells, which are reminiscent of bacterial pilli, have been recently rediscovered (Köhler et al. 1997). ...
Plants have unique biological attributes of great interest to researchers investigating population dynamics. Yet, until recently, organelle DNA had been seldom utilized for phylogeographic studies in plants. While cpDNA variation has been used extensively to construct interspecific phylogenies, many researchers have considered that the relatively low levels of intraspecific variation revealed by early studies of plant organelle DNA render these genomes of little use for intraspecific studies. In this review we introduce the field of plant phylogeography based on organelle polymorphisms by providing a detailed discussion of the processes underlying this variation. Progress in molecular organelle genetics has provided insights into the structure, variation, inheritance, vegetative segregation and recombination of organelle genomes. While some of these features (e.g. low substitution rates) may complicate phylogeographic studies, others (e.g. presence of two genomes and frequency of atypical modes of transmission) offer unique opportunities, many of which are virtually unexplored.
... The fact that plastids rarely fuse (Sears 1980) must limit the effective number of plastid genomes per cell. The observation that rare cases of plastid fusion seem to account for unusually slow sorting out (Vaughn 1981; Lax et al. 1987 ) supports this view. Transient tubular connections between chloroplasts within cells, which are reminiscent of bacterial pilli, have been recently rediscovered (Köhler et al. 1997). ...
Plants have unique biological attributes of great interest to researchers investigating population dynamics. Yet, until recently, organelle DNA had been seldom utilized for phylogeographic studies in plants. While cpDNA variation has been used extensively to construct interspecific phylogenies, many researchers have considered that the relatively low levels of intraspecificvariation revealed by early studies of plant organelle DNA render these genomes of little use for intraspecific studies. In this review we introduce the field of plant hylogeography based on organelle polymorphisms by providing a detailed discussion of the processes underlying this variation. Progress in molecular organelle genetics has provided insights into the structure, variation,inheritance, vegetative segregation and recombination of organelle genomes. While some of these features (e.g. low substitution rates) may complicate phylogeographic studies, others(e.g. presence of two genomes and frequency of atypical modes of transmission) offer unique opportunities, many of which are virtually unexplored
... Lee et al. (1988) and Johnson and Palmer (1989) demonstrated the existence of biparental inheritance and heteroplasmic cells with both chlorophyll-deficient and wild-type plastids in Medicago. In other cases, such as Gossypium (Lax et al. 1987), where uniparental inheritance occurs, heteroplasmy can occur when segregation of wildtype and mutant plastids is incomplete and heteroplasmic gametes are produced. Persistent plastid heteroplasmy can be transmitted by slow vegetative segregation as found by Gillham et al. (1991). ...
Ribosomal genes are considered to have a high degree of sequence conservation between species and also at higher taxonomic levels. In this paper we document a case where a single individual of Cynomorium coccineum (Cynomoriaceae), a nonphotosynthetic holoparasitic plant, contains highly divergent plastid ribosomal genes. PCR amplification a nearly complete ribosomal DNA cistron was performed using genomic DNA, the products cloned, and the 23S rDNA genes were sequenced from 19 colonies. Of these, five distinct types were identified. Fifteen of the sequences were nearly identical (11 or fewer differences) and these were designated Type I. The remaining types (II-V) were each represented by a single clone and differed from Type I by 93 to 255 changes. Compared with green vascular plants, we found that there are more substitutional differences in the 23S rDNA sequences within a single individual of Cynomorium than among all sequenced photosynthetic vascular plants. Several trends of molecular evolution observed in 16S rDNA from other holoparasitic angiosperms and heterotrophic green algae have been also observed in Cynomorium 23S rDNA. Higher-order structures were constructed for representatives of the five clone types, and in all cases these possessed complete complements of the major structural elements present in functional plastid 23S rRNAs. These data indicate that such molecules may be subject to purifying selection, thus providing indirect evidence that they have retained some degree of functionality. This intraindividual polymorphism is probably a case of plastid heteroplasmy but translocation of ribosomal cistrons to the nucleus or mitochondria has not been tested and therefore cannot be ruled out.
... All seeds derived from self-pollinated chloroplast transgenic plants germinated on kanamycin and therefore, no Mendelian segregation was observed in the tested seeds. Uniparental maternal inheritance of cotton plastid genome has been reported earlier where the mechanism of maternal inheritance was investigated in depth and established (Lax et al., 1987; Hagemann, 2004). ...
Chloroplast genetic engineering overcomes concerns of gene containment, low levels of transgene expression, gene silencing, positional and pleiotropic effects or presence of vector sequences in transformed genomes. Several therapeutic proteins and agronomic traits have been highly expressed via the tobacco chloroplast genome but extending this concept to important crops has been a major challenge; lack of 100 homologous species-specific chloroplast transformation vectors containing suitable selectable markers, ability to regulate transgene expression in developing plastids and inadequate tissue culture systems via somatic embryogenesis are major challenges. We employed a 'Double Gene/Single Selection (DGSS)' plastid transformation vector that harbors two selectable marker genes (aph A-6 and npt II) to detoxify the same antibiotic by two enzymes, irrespective of the type of tissues or plastids; by combining this with an efficient regeneration system via somatic embryogenesis, cotton plastid transformation was achieved for the first time. The DGSS transformation vector is at least 8-fold (1 event/2.4 bombarded plates) more efficient than 'Single Gene/Single Selection (SGSS)' vector (aph A-6; 1 event per 20 bombarded plates). Chloroplast transgenic lines were fertile, flowered and set seeds similar to untransformed plants. Transgenes stably integrated into the cotton chloroplast genome were maternally inherited and were not transmitted via pollen when out-crossed with untransformed female plants. Cotton is one of the most important genetically modified crops (120 billion US dollars US annual economy). Successful transformation of the chloroplast genome should address concerns about transgene escape, insects developing resistance, inadequate insect control and promote public acceptance of genetically modified cotton.
... Parker' reacted sequentially with anti-RuBisCo, goat anti-rabbit labeled with peroxidase, and a peroxidase incubation media resulted in strong immunostaining of the large subunit of RuBisCo (Fig. 7). Like other anti-RuBisCo sera prepared to the holoenzyme (3), this anti-tobacco RuBisCo (5) recognizes only the large subunit. Although Zemel and Gepstein (17) found that protease inhibitors were required to preserve guard cell RuBisCo, the relatively simple homogenization media used in this report preserved an apparently unaltered large subunit of RuBisCo. ...
Two immunological approaches were used to determine if ribulose bisphosphate carboxylase oxygenase (RuBisCo) is present in guard cell chloroplasts. Immunocytochemistry on thin plastic sections using tissue samples that were processed using traditional glutaraldehyde/osmium fixation and then restored to antigenicity with metaperiodate treatment, resulted in labeling over wild-type mesophyll and guard cell plastids of several green and white variegated Pelargonium chimeras. The density of immunogold labeling in guard cell chloroplasts was only about one-seventh of that noted in mesophyll chloroplasts on a square micron basis. Because guard cell chloroplasts are much smaller than mesophyll chloroplasts, and occur at lower quantities/cell, the relative differences in RuBisCo concentration between the cell types indicate that guard cells have only 0.48% of the RuBisCo of mesophyll cells. No reaction was noted over 70S ribosomeless plastids of these chimeras even though adjacent green chloroplasts were heavily stained, indicating the high specificity of the reaction for RuBisCo. Spurr's resin gave the most successful colloidal gold labeling in terms of low background staining and structural detail but L. R. White's resin appeared to be superior for antigen retention. In the white leaf edges of the white and green Pelargonium chimeras, the only green, functional chloroplasts are in the guard cells. When either whole tissue or plastid enriched extracts from this white tissue were electrophoresed, blotted, and probed with anti-RuBisCo a large subunit band was detected, identical to that in the green tissue. These data indicate that a low, but detectable, level of RuBisCo is present in guard cell chloroplasts.
Der Ursprung, die Entwicklung und die Formierung von Laubblatt-Mustern konnten bei ausgewählten variegaten Formen der Araceae, Asteraceae, Ericaceae, Maranthaceae und Rosaceae erklärt werden. Die Pflanzen wurden je nach Problematik untersucht und in drei verschiedene Gruppen verteilt: In der ersten Gruppe, Blattmuster mit unregelmäßiger makulater Musterung, Monstera deliciosa, Syngonium podophyllum und die Sorten 'Pirol' und 'Luyona' von Dendranthema grandiflorum zeigen ein unregelmäßiges Laubblattmuster und keine weiß-randigen bzw. weißkernigen Periklinalchimären. Mischzellen wurden durch direkte (mikroskopisch) und indirekte (In-vitro-Kultur und Selbstungen) Nach der Plastidenentmischung in den Schichten des Sprossmeristems wurde bei Syngonium, Monstera und den zwei Sorten von Dendranthema die GW-Form als einzige stabile Periklinalchimäre nachgewiesen. In der zweiten Gruppe, Immerspaltende Periklinalchimären, die chimärische Konstitution GA bei Spiraea bumalda 'Goldflame' konnte durch Wurzelaustriebe (BATESON-Test) und die Adventivsprossinduktion aus Kallus nachgewiesen werden. Darüber hinaus konnten mehrfach perikline Aufspaltungen der ersten Sprossscheitelschicht (Reduplikation von L1) nachgewiesen werden, die zur Entstehung der Panaschierung von 'Goldflame' zur Entmischung führten. Bei den Untersuchungen der In-vitro-Regenerate aus der Kalluskultur und der Wurzelaustriebe an S. bumalda 'Shirobana' wurde festgestellt, dass diese Pflanze keine Chimäre ist und das auftretende Muster der Blüten genetisch kontrolliert ist. In der dritten Gruppe, Hypoderm und Beeinflussung der Musterbildung: Die unmaskierten Binnenfelder bei Ctenanthe lubbersiana 'Variegata' und der Rhododendron-Hybride 'Goldflimmer' sind durch die Existenz eines Hypoderms zu erklären. Bei Ctenanthe lubbersiana 'Variegata' befindet sich ein Hypoderm an der Blattoberseite und der Blattunterseite. Bei 'Goldflimmer' liegt nur unter der oberen Epidermis ein einschichtiges Hypoderm vor. Infolgedessen fehlt an der Blattoberseite die Maskierung Das gelbe Binnenfeld des Blattmusters ist durch eine grüne Mesophyllschicht unterlagert.
Harris, S. A. & Ingram, R.: Chloroplast DNA and biosystematics: The effects of intraspecific diversity and plastid transmission. ‐ Taxon 40: 393–412. 1991. ‐ ISSN 0040‐0262.
The widespread application of chloroplast DNA (cpDNA) to plant biosystematics is usually based on a number of assumptions. The assumption of low levels of intraspecific cpDNA variation is reviewed and the conclusion reached that far from being rare, intraspecific cpDNA variation is relatively common. The evidence for the assumption of predominantly maternal plastid transmission is also reviewed. In both of the cases some of the possible effects on phylogenetic reconstruction are considered.
SUMMARYA nuclear-gene mutation of the C3 grass Lolium temulentum L., which arose following cell suspension culture and plant regeneration, is manifested as delayed and incomplete greening, which occurs from the leaf tip downwards. Many plastids in the mutant exhibit abnormal morphology when examined by transmission electron microscopy; the plastid outer envelope lacks integrity and thylakoids, while still stacked, are spread over a wide area surrounded by diffuse stromal contents. These aberrant plastids can coexist with apparently normal chloroplasts in the same cell of mutant plants. Levels of chlorophyll a and b, and carotenoids, are all lower in the mutants than in normal Lolium temulentum. Leaf length, absolute growth rate, and number of cells per unit length at the leaf base, are greatly reduced (20-30% the normal values) in slow-to-green plants, but relative growth rate, duration of leaf growth, length of cell division zone and proportion of cells dividing are little affected. This novel mutant is a potentially valuable resource for studying interrelationships between photosynthetic function and leaf extension growth in grasses.
Summary Many of the studies of chloroplast ontogeny in higher plants have utilized suboptimal conditions of light and growth to assess development. In this study, we utilized structural, immunological, and physiological techniques to examine the development of the chloroplast in fieldgrown cotton (Gossypium hirsutum cv. “MD 51 ne”). Our youngest leaf sample developmentally was completely folded upon itself and about 0.5 cm in length; leaves of this same plastochron were followed for three weeks to the fully expanded leaf. The chloroplasts at the earliest stage monitored had almost all of the lamellae in small, relatively electron-opaque grana, with relatively few thylakoids which were not appressed on at least one surface. During the development of the thylakoids, the membranes increase in complexity, with considerable stroma lamellae development and an increase in the number of thylakoids per granum. Besides the increase in complexity, both the size and numbers of the chloroplast increase during the development of the leaf. Developmental changes in six thylakoid proteins, five stromal proteins, and one peroxisomal protein were monitored by quantitative immunocytochemistry. Even at the earliest stages of development, the plastids are equipped with the proteins required to carry out both light and dark reactions of photosynthesis. Several of the proteins follow three phases of accumulation: a relatively high density at early stages, a linear increase to keep step with chloroplast growth, and a final accumulation in the mature chloroplast. Photosystem-II(PS II)-related proteins are present at their highest densities early in development, with an accumulation of other parts of the photosynthetic apparatus at a latter stage. The early accumulation of PS-II-related proteins correlates with the much lower ratio of chlorophylla tob in the younger leaves and with the changes in fluorescence transients. These data indicate that some of the conclusions on chloroplast development based upon studies of intercalary meristems of monocots or the greening of etiolated plants may not be adequate to explain development of chloroplasts in leaves from apical meristems grown under natural conditions.
Effects of paraquat were monitored by electron microscopic, cytochemical, and in vivo spectrophotometric procedures. Electron microscopy of paraquat-treated pea leaf discs indicates that the chloroplasts are affected primarily with both thylakoid dilation and apparent lamellar fusions, resulting in a ‘honeycomb’ of lamellae. No ultra-structural effects were noted when leaf discs were incubated in both DCMU and paraquat. Paraquat-induced peroxide was located cytochemically along the stroma lamellae, on the ends of the grana stack and in the dilated thylakoids. Less destruction was noted in those samples where peroxide was precipitated by cerium chloride, indicating that peroxide or one of its products is one of the major causes of paraquat toxicily. In C4 plants mesophyll plastids exhibited much more peroxide deposition than was detected in bundle sheath plastids. No plastid peroxide depositions were noted in a photobleaching mutant that is resistant to paraquat. In situ cytochrome f oxidation-reduction was followed during paraquat treatments and resulted in a hastening of the dark re-reduction of cytochrome f after only 1 h of treatment. This effect was prevented by DCMU or CeCI3. Electron transport, as measured by cytochrome f oxidation-reduction, was completely obliterated by a 24 h paraquat treatment. These in situ studies on paraquat confirm previous studies that the primary effect in the light is on the plastid and involves peroxide or further products generated by peroxide.