Plastid DNA diversity in natural populations of Beta maritima showing additional variation in sexual phenotype and mitochondrial DNA

Summary Plants of two natural populations of Beta maritima, characterized by high percentages of male-sterile plants, have been investigated for organelle DNA polymorphism. We confirm the two classes of mitochondrial DNA variation previously described: (i) mitochondrial DNA (mtDNA) type N is associated with male fertility, whereas mtDNA type S can cause cytoplasmic male sterility (CMS); (ii) the 10.4-kb linear plasmid is observed in both types of mitochondria and is not correlated with the cytoplasmic male sterility occurring in this plant material. A third polymorphism is now described for chloroplast DNA (ctDNA). This polymorphism occurs within single populations of Beta maritima. Three different ctDNA types have been identified by HindIII restriction analysis. Among the plants studied, ctDNA type 1 is associated with N mitochondria and type 2 with S mitochondria. Chloroplast DNA type 3 has been found both in a fertile N plant and in a sterile S plant. This finding suggests that the chloroplast DNA polymorphism reported is not involved in the expression of male sterility. A comparison with Beta vulgaris indicates that ctDNA type 3 of Beta maritima corresponds to the ctDNA of fertile sugar beet maintainer lines. The three types of Beta maritima ctDNA described in this study differ from the ctDNA of male-sterile sugar beet.     

A rapid assay for chloroplast-encoded triazine resistance in higher plants

We have designed a simple and rapid assay for chloroplast-based triazine resistance in higher plants using PCR amplification of thepsbA gene coupled toMaeI digestion of the amplified product to distinguish triazine resistant from sensitive biotypes. Our assay is universal and avoids the need of lengthy procedures of previously published assays, which either required spraying of seedlings in a controlled environment, quantification of chlorophyll fluorescence of leaf discs after incubation in triazine solution, DNA sequencing of thepsbA gene, or Southern-blot analysis. Our diagnostic system is qualitative, reliable, fast and simple. More than 100 seedlings taken directly from the field can be analyzed in one day. This system has a direct application towards a more rational use of herbicides in production fields. It also represents a valuable tool to monitor spreading of resistant biotypes through time and space and can serve as a model system applicable to other gene monitoring needs.     

An in vitro molecular study of the Nicotiana tabacum L. genome in the presence or absence of the herbicide atrazine

  Nicotiana tabacum L. somaclones both selected and not selected for tolerance to the triazine herbicide atrazine were used to compare tissue culture-induced variability in the presence or absence of stress. Two types of repeated sequences (rDNA and a randomly cloned, anonymous sequence) were analysed both qualitatively and quantitatively, and overall genome variation was assessed by RAPDs. Multiplicity differences were found for the two sequences both between the tolerant and susceptible group and within each group with respect to leaf DNA, but no qualitative differences were detected with either RFLPs or RAPDs. Moreover, we investigated whether stressinduced variation in the atrazine target gene, the chloroplast psbA gene, was responsible for herbicide tolerance by analysing two possible resistance mechanisms: the presence of a specific point mutation in the gene and its amplification and/or increased expression. Some somaclones were shown to be a mosaic for psbA gene mutation, but the number of cells or plastid genomes involved seemed too low to account for tolerance in the whole tissue. Atrazine tolerance could then be due to an increase in the number of plastids/plastid genomes or/and to a permanent response to respiration inhibition whose basis is, up to now, unknown. Received: 18 July 1997/Accepted: 22 August 1997     

A Ser–Gly substitution in plastid-encoded photosystem II D1 protein is responsible for atrazine resistance in foxtail millet (<Emphasis Type="Italic">Setaria italica</Emphasis>)

A foxtail millet (Setaria italica L. Beauv.) line resistant to atrazine was obtained through interspecific hybridization between wild S. viridis L. Beauv. and cultivated S. italica. The resistance was proved to be controlled by a chloroplast-inherited gene and it has further been utilized in foxtail millet production. However, the sequence information of the putative atrazine resistance gene, psbA in foxtail millet’s chloroplast genome encoding photosystem II D1 protein (32 kDa thylakoid membrane protein) (photosystem Q_B protein) and the mutation site responsible for the resistance are not known. In this paper the psbA sequences of six atrazine susceptible/resistant foxtail millet varieties were obtained and compared. The results indicated that there was only one amino acid difference between susceptible and resistance gene, resulting from a single base substitution. It was concluded that a mutant allele of photosystem II protein D1 encoding a Gly residue instead of a Ser residue at position 264 is a major gene of resistance to atrazine. Moreover, the phylogenetic tree based on the psbA coding region of thirty-five plant species was carried out. The phylogenetic relationship between S. italica and other plants and the related evolutionary issues were discussed and it was suggested that psbA sequences could be used in phylogenetic studies in plants. Xiaoping Jia and Jincheng Yuan have equal contribution.     

Demographic History of Shorea curtisii (Dipterocarpaceae) Inferred from Chloroplast DNA Sequence Variations

We assessed the variability of chloroplast DNA sequences in populations of the dipterocarp forest tree, Shorea curtisii. This species is widely distributed in hill and coastal hill dipterocarp forests of the Malay Peninsula, whereas isolated populations are found in the coastal hills of north Borneo. Two chloroplast DNA regions (1555 bp of trnH‐psbA‐trnK and 925 bp of trnL‐trnF) were sequenced from 123 individuals collected from six Malay Peninsula and two Bornean populations. There were 15 chloroplast haplotypes derived from 16 polymorphic sites. A haplotype network revealed two distinct haplogroups that correlate with two geographic regions, the Malay Peninsula and Borneo. These two haplogroups differed by a number of mutations, and no haplotypes were shared between populations from the different geographic regions. This suggests an ancient diversification of these haplogroups, and that long‐distance seed dispersal was unlikely to have occurred during the Pleistocene when the Sunda Shelf was a contiguous landmass. Phylogenetic analysis of the haplotypes together with those found in other Shorea species showed that two haplogroups in S. curtisii appear in different positions of the phylogenetic tree. This could be explained by the persistence of ancestral polymorphisms or by ancient chloroplast capture. Low levels of genetic differentiation were found between populations within each geographic region. Signature of a bottleneck followed by demographic expansion was detected in the Malay Peninsula haplogroup. The presence of two distinct evolutionary lineages in the different regions suggests that they should be managed independently to conserve the major sources of genetic diversity in S. curtisii.     

Ribosome-binding sites on chloroplastrbcL andpsbA mRNAs and light-induced initiation of D1 translation

Chloroplast ribosome-binding sites were identified on the plastidrbcL andpsbA mRNAs using toeprint analysis. TherbcL translation initiation domain is highly conserved and contains a prokaryotic Shine-Dalgarno (SD) sequence (GGAGG) located 4 to 12 nucleotides upstream of the initiator AUG. Toeprint analysis ofrbcL mRNA associated with plastid polysomes revealed strong toeprint signals 15 nucleotides downstream from the AUG indicating ribosome binding at the translation initiation site.Escherichia coli 30S ribosomes generated similar toeprint signals when mixed withrbcL mRNA in the presence of initiator tRNA. These results indicate that plastid SD sequences are functional in chloroplast translation initiation. ThepsbA initiator region lacks a SD sequence within 12 nucleotides of the initiator AUG. However, toeprint analysis of soluble and membrane polysome-associatedpsbA mRNA revealed ribosomes bound to the initiator region.E. coli 30S ribosomes did not associate with thepsbA translation initiation region.E. coli and chloroplast ribosomes bind to an upstream region which contains a conserved SD-like sequence. Therefore, translation initiation onpsbA mRNA may involve the transient binding of chloroplast ribosomes to this upstream SD-like sequence followed by scanning to localize the initiator AUG. Illumination 8-day-old dark-grown barley seedlings caused an increase in polysome-associatedpsbA mRNA and the abundance of initiation complexes bound topsbA mRNA. These results demonstrate that light modulates D1 translation initiation in plastids of older dark-grown barley seedlings.     

Phylogeny of Ultra-Rapidly Evolving Dinoflagellate Chloroplast Genes: A Possible Common Origin for Sporozoan and Dinoflagellate Plastids

Complete chloroplast 23S rRNA and psbA genes from five peridinin-containing dinoflagellates (Heterocapsa pygmaea, Heterocapsa niei, Heterocapsa rotun-data, Amphidinium carterae, and Protoceratium reticulatum) were amplified by PCR and sequenced; partial sequences were obtained from Thoracosphaera heimii and Scrippsiella trochoidea. Comparison with chloroplast 23S rRNA and psbA genes of other organisms shows that dinoflagellate chloroplast genes are the most divergent and rapidly evolving of all. Quartet puzzling, maximum likelihood, maximum parsimony, neighbor joining, and LogDet trees were constructed. Intersite rate variation and invariant sites were allowed for with quartet puzzling and neighbor joining. All psbA and 23S rRNA trees showed peridinin-containing dinoflagellate chloroplasts as monophyletic. In psbA trees they are related to those of chromists and red algae. In 23S rRNA trees, dinoflagellates are always the sisters of Sporozoa (apicomplexans); maximum likelihood analysis of Heterocapsa triquetra 16S rRNA also groups the dinoflagellate and sporozoan sequences, but the other methods were inconsistent. Thus, dinoflagellate chloroplasts may actually be related to sporozoan plastids, but the possibility of reproducible long-branch artifacts cannot be strongly ruled out. The results for all three genes fit the idea that dinoflagellate chloroplasts originated from red algae by a secondary endosymbiosis, possibly the same one as for chromists and Sporozoa. The marked disagreement between 16S rRNA trees using different phylogenetic algorithms indicates that this is a rather poor molecule for elucidating overall chloroplast phylogeny. We discuss possible reasons why both plastid and mitochondrial genomes of alveolates (Dinozoa, Sporozoa and Ciliophora) have ultra-rapid substitution rates and a proneness to unique genomic rearrangements. Received: 27 December 1999 / Accepted: 24 March 2000     

Comparison of chloroplast and mitochondrial DNA from five morphologically distinct Beta vulgaris cultivars: sugar beet,fodder beet,beet root,foliage beet,and Swiss chard

Summary Two cytoplasms, N and S, are used in the breeding of sugar beet, Beta vulgaris var. altissima. These cytoplasms can be distinguished by their mitochondrial DNA. In an attempt to detect new cytoplasms, we compared the restriction profiles of chloroplast and mitochondrial DNA from five different cultivars of Beta vulgaris. All restriction patterns of chloroplast DNA were identical. With the exception of sugar beet with S-cytoplasm, all cultivars studied showed the same restriction profile of mitochondrial DNA, indicating that these cultivars all contain the N-cytoplasm. These results are discussed with regard to the large morphological differences of the cultivars and the cytoplasmic variability found in natural populations of the wild beet, Beta maritima.     

Construction of a Chlamydomonas reinhardtii mutant with an intronless psbA gene

Efficient chloroplast transformation systems now available allow the manipulation of the evolutionarily highly conserved psbA gene in the eucaryotic organism Chlamydomonas reinhardtii. Two copies of this gene in the inverted repeat region of the chloroplast genome contain four large group I introns. To analyse possible functions of these introns and to generate a mutant for simplified psbA gene manipulations, a psbA cDNA fragment was introduced into a psbA deletion mutant using the biolistic transformation method. A transformant with no introns in the psbA gene has been obtained and represents the first example of the removal of a complete set of introns from a chloroplast gene. The newly generated strain is photosynthetically competent and contains no detectable recipient genome copies. The loss of all four introns appears to be phenotypically silent.     

A regression analysis of the summer population dynamics of Polyarthra vulgaris in a northern Michigan bog lake

The population dynamics of a planktonic rotifer (Polyarthra vulgaris) were examined in a brown water, acid lake in northern Michigan, U.S.A. Predation by Chaoborus punctipennis and low food (Navicula spp. and Cyclotella spp.) concentrations were the main factors limiting P. vulgaris populations of all factors examined. The data presented here support a hypothesis for zooplankton limitation by an invertebrate predator.     

Preservation of Solanum pimpinellifolium genomic fragments in recombinant genotypes improved the fruit quality of tomato

Five recombinant inbred lines obtained from the F₂ generation of an interspecific cross between cultivar, Caimanta (Cai, Solanum lycopersicum) and wild accession, LA722 (P, S. pimpinellifolium) were crossed to obtain the second cycle hybrids (SCH). Eleven fruit quality traits were assessed in evaluating phenotypic variability among genotypes P, Cai, F₁ (Cai × P), five RILs, and 10 SCH. One of the five recombinant inbred lines and three SCH had higher values than P, as the best genotype for shelf life. Sequence-related amplified polymorphism was used as the molecular method for detecting polymorphism among these 18 genotypes. The percentage of polymorphism in RILs and SCH was 61% and 66% respectively. Moreover, some bands detected in P were present in SCH. Several multivariate analyses were performed to find agreement between the phenotypic variability observed for fruit quality traits and the polymorphism obtained from sequence-related amplified polymorphism markers. A general Procrustes analysis estimated that there was a consensus proportion of 75% between phenotypic and molecular data. There was considerable preservation of some bands from the wild genotype, which could increase the variability in fruit quality traits in populations where the genetic diversity is limited.     

Functional significance of triazine-herbicide resistance in defence of Senecio vulgaris against a rust fungus

We investigated the functional significance of plant performance (dry mass, photosynthesis) in plant defence (resistance and tolerance) against pathogen infection, and potential negative cross-resistance between herbicide resistance and plant defence against disease. We compared isonuclear triazine-herbicide-resistant (TR) and -susceptible (TS) biotypes of Senecio vulgaris, in the presence and absence of infection by the rust Puccinia lagenophorae. In a growth chamber study with two reduced irradiance levels, rust infection had a severe effect on plant performance with infected plants having 55% less dry mass and 54% reduced whole-plant photosynthesis than non-infected plants. The TR biotype was more susceptible (reduced resistance) to the pathogen, but the biotypes did not differ in their ability to compensate for rust infection (tolerance). TR plants were less productive than TS plants when grown non-shaded (ca. 10% full sunlight) but not when shaded (ca. 5% full sunlight). This is especially important for situations, where S. vulgaris grows under the crop canopy (e.g. in maize). Here, very low light levels might contribute to a numerical increase of TR relative to TS plants even when only occasionally treated with triazine. Whole-plant photosynthesis was reduced by 21% in TR plants as compared to the TS biotype, and by 59% in plants grown in the shaded as compared to the non-shaded treatment. When whole-plant photosynthesis values were corrected for the estimated leaf area of plants, we found no significant variation between biotypes, shade treatments or rust treatments. In experimental mixed TR:TS field populations, the proportion of TR plants decreased more rapidly in rust-infected populations than uninfected. This finding, together with the lower resistance in the TR than the TS biotype to the rust fungus observed in the growth chamber experiment, may indicate negative cross-resistance, which is a potential tool in the management of herbicide-resistant weeds.     

Chloroplast and nuclear DNA variation among homozygous plants in a population of the autogamous annualMicroseris douglasii (Asteraceae,Lactuceae)

The autogamous diploid annualMicroseris douglasii of California occurs in many isolated populations. The populations consist of one to many highly inbred biotypes. Morphological variation among populations usually is greater than within populations. In spite of the virtual absence of gene flow even within populations, genetically determined character differences are randomly distributed and associated throughout the range of the species. Recent evidence even suggests introgression of chloroplasts from the relatedM. bigelovii. Offspring families from 25 plants of a very variable population were raised and examined for segregation of morphological and molecular (RAPD) markers. All 25 original plants were completely homozygous for all markers, but each differed from all others at least in some markers. The population consisted of two genetically isolated groups of plants: a distinct inbred line (3 plants) and 22 plants with random associations of a common set of markers and characters, possibly recombinant inbreds from a past hybridization event. One of these 22 plants contained a chloroplast genome found inM. bigelovii, the other 24 plants a chloroplast genome found only inM. douglasii.     

Hybrid swarms: catalysts for multiple evolutionary events in Senecio in the British Isles

Background: Introgressive hybridisation is an evolutionary catalyst producing novel variants able to explore new ecological niches and evolve as new hybrid taxa. However, the role of ‘hybrid swarms’ – highly variable populations produced following interspecific hybridisation – in generating this evolutionary novelty has been poorly studied.

Aims: We examine the alternative origins of tetraploid hybrid derivatives of Senecio vulgaris and S. squalidus, via local polytopic formation or long-distance dispersal from a single perennial hybrid swarm around Cork, Ireland.

Methods: Morphometric, isozyme and chloroplast DNA analysis.

Results: The Cork hybrid swarm and UK hybrid swarms exhibited a broad range of morphological variation and contained individuals similar to the stable tetraploid hybrid derivatives; S. eboracensis and S. vulgaris var. hibernicus. Chloroplast DNA analysis shows that S. eboracensis did not evolve from the Cork hybrid swarm. However, UK S. vulgaris var. hibernicus populations exhibit a broad range of variation for both chloroplast and isozyme markers, but were not distinguishable from Cork material.

Conclusions: Our study confirms that S. eboracensis did not evolve from the Cork hybrid swarm, and while our analyses could not demonstrate this conclusively for S. vulgaris var. hibernicus the ease with which hybrid swarms have been generated in the past makes a polytopic origin for S. vulgaris var. hibernicus the most likely scenario.     

Chloroplast DNA of black pine retains a residual inverted repeat lacking rRNA genes: nucleotide sequences of trnQ, trnK, psbA, trnI and trnH and the absence of rps16

Summary A physical map of black pine (Pinus thunbergii) chloroplast DNA (120 kb) was constructed and two separate portions of its nucleotide sequence were determined. One portion contains trnQ-UUG, ORF510, ORF83, trnK-UUU (ORF515 in the trnK intron), ORF22, psbA, trnI-CAU (on the opposing strand) and trnH-GUG, in that order. Sequence analysis of another portion revealed the presence of a 495 by inverted repeat containing trnI-CAU and the 3 end of psbA but lacking rRNA genes. The position of trnI-CAU is unique because most chloroplast DNAs have no gene between psbA and trnH (trnI-CAU is usually located further downstream). Black pine chloroplast DNA lacks rps16, which has been found between trnQ and trnK in angiosperm chloroplast DNAs, but possesses ORF510 instead. This ORF is highly homologous to ORF513 found in the corresponding region of liverwort chloroplast DNA and ORF563 located downstream from trnT in Chlamydomonas moewusii chloroplast DNA. A possible pathway for the evolution of black pine chloroplast DNA is discussed.     

Knockdown of the Arabidopsis thaliana chloroplast protein disulfide isomerase 6 results in reduced levels of photoinhibition and increased D1 synthesis in high light

A chloroplast protein disulfide isomerase (PDI) was previously proposed to regulate translation of the unicellular green alga Chlamydomonas reinhardtii chloroplast psbA mRNA, encoding the D1 protein, in response to light. Here we show that AtPDI6, one of 13 Arabidopsis thaliana PDI genes, also plays a role in the chloroplast. We found that AtPDI6 is targeted and localized to the chloroplast. Interestingly, AtPDI6 knockdown plants displayed higher resistance to photoinhibition than wild‐type plants when exposed to a tenfold increase in light intensity. The AtPDI6 knockdown plants also displayed a higher rate of D1 synthesis under a similar light intensity. The increased resistance to photoinhibition may not be rationalized by changes in antenna or non‐photochemical quenching. Thus, the increased D1 synthesis rate, which may result in a larger proportion of active D1 under light stress, may led to the decrease in photoinhibition. These results suggest that, although the D1 synthesis rates observed in wild‐type plants under high light intensities are elevated, repair can potentially occur faster. The findings implicate AtPDI6 as an attenuator of D1 synthesis, modulating photoinhibition in a light‐regulated manner.     

Quantitative assessment of heteroplasmy levels in <Emphasis Type="Italic">Senecio vulgaris</Emphasis> chloroplast DNA

Heteroplasmy in coding chloroplast DNA was only recently shown to occur and was so far not quantitatively assessed. We present a quantitative analysis of cpDNA heteroplasmy levels at a triazine-resistance determining site within and between individual Senecio vulgaris plants. Detectable levels of heteroplasmic haplotypes were observed in all tested plants. As expected, the levels of heteroplasmy vary greatly between plants. However, even within individual plants, the fraction of one haplotype may cover a range from below 1 to well over 90. Our results suggest that heteroplasmy may be a common phenomenon in S. vulgaris.Possible consequences for molecular diagnostics of chloroplast encoded traits as well as evolutionary consequences of chloroplast heteroplasmy are discussed.     

Nucleotide sequence of the chloroplast gene responsible for triazine resistance in canola

Summary The nucleotide sequence for the psbA gene from a triazine resistant cultivar of B. napus (cv Triton) has been determined. This gene encodes an open reading frame of 353 amino acids that is highly homologous to other higher plant psbA genes at both the nucleotide and amino acid levels. As has been found for other triazine resistant psbA genes, the Triton psbA contains an A to G nucleotide change which results in a serine to glycine amino acid substitution at position 264. The B. napus psbA gene also has a G insertion at position –9 resulting in a ribosome binding site sequence (AGGA) just before the initial methionine and suggesting that the entire open reading frame is translated. A large (72 bp) insertion is also found upstream of the B. napus psbA gene which resembles a similar insertion in the mustard psbA. The uncloneable nature of the entire gene is further investigated through reconstruction experiments and the implications discussed.