Plastid genome characterisation in Brassica and Brassicaceae using a new set of nine SSRs.

We report a new set of nine primer pairs specifically developed for amplification of Brassica plastid SSR markers. The wide utility of these markers is demonstrated for haplotype identification and detection of polymorphism in B. napus, B. nigra, B. oleracea, B. rapa and in related genera Arabidopsis, Camelina, Raphanus and Sinapis. Eleven gene regions (ndhB-rps7 spacer, rbcL-accD spacer, rpl16 intron, rps16 intron, atpB-rbcL spacer, trnE-trnT spacer, trnL intron, trnL-trnF spacer, trnM-atpE spacer, trnR-rpoC2 spacer, ycf3-psaA spacer) were sequenced from a range of Brassica and related genera for SSR detection and primer design. Other sequences were obtained from GenBank/EMBL. Eight out of nine selected SSR loci showed polymorphism when amplified using the new primers and a combined analysis detected variation within and between Brassica species, with the number of alleles detected per locus ranging from 5 (loci MF-6, MF-1) to 11 (locus MF-7). The combined SSR data were used in a neighbour-joining analysis (SMM, D (DM) distances) to group the samples based on the presence and absence of alleles. The analysis was generally able to separate plastid types into taxon-specific groups. Multi-allelic haplotypes were plotted onto the neighbour joining tree. A total number of 28 haplotypes were detected and these differentiated 22 of the 41 accessions screened from all other accessions. None of these haplotypes was shared by more than one species and some were not characteristic of their predicted type. We interpret our results with respect to taxon differentiation, hybridisation and introgression patterns relating to the 'Triangle of U'.

Direct gene transfer in potato: a comparison of particle bombardment of leaf explants and PEG-mediated transformation of protoplasts.

Direct gene transfer methods in potato would facilitate the transfer of multiple genes and the manipulation of metabolic pathways in this species. In this study, up to 1.8 transformation events per shot (=0.5 per bombarded leaf) and 67.2 events per million protoplasts treated were obtained with particle bombardment and PEG-mediated direct DNA uptake, respectively. Limited disassociation of both HPT and GUS genes appeared to occur during the process of integration in only 19% of transformants. A large number of transformed potato plants with transgene expression at levels comparable to Agrobacterium-mediated transformation was obtained. High levels of GUS expression were only obtained in lines derived from PEG treatment. No correlation between the number of gene insertions and gene expression levels was found, suggesting that multiple insertions may have little or no effect on transgene expression.

NAN fusions: a synthetic sialidase reporter gene as a sensitive and versatile partner for GUS.

GUS continues to be the reporter of choice for many gene fusion applications, due to the unparalleled sensitivity of the encoded enzyme and the ease with which it can be quantified in cell-free extracts and visualized histochemically in cells and tissues. A compatible and functionally equivalent reporter gene would facilitate dual promoter studies and internal standardization of expression analyses in the same plant. A search for a candidate enzyme activity not found in plants, which might form the basis of a novel GUS-compatible reporter system, led us to investigate nanH, a Clostridium perfringens gene which encodes the so-called 'small' cytoplasmic sialidase. Expression of the native, AT-rich nanH gene in transgenic plants did not, however, result in detectable sialidase activity. For this reason, a codon-optimized derivative, NAN, was synthesized which possesses a GC content similar to that found in highly expressed plant genes. NAN enzyme activity was expressed at high levels in both stably and transiently transformed cells, possessed kinetic and stability properties similar to those of GUS, and showed optimal activity in GUS buffer. Moreover, NAN and GUS activity could be visualized simultaneously in polyacrylamide gels using the corresponding methylumbelliferone-based substrates, and in whole seedlings and tissue sections using the histochemical substrates 5-bromo-4-chloro-3-indolyl alpha-d-N-acetylneuraminic acid (X-NeuNAc) and 5-bromo-6-chloro-3-indolyl beta-d-glucuronide (X-GlucM), respectively.

Tissue-specific and developmental-specific expression of an Arabidopsis thaliana gene encoding the lipoamide dehydrogenase component of the plastid pyruvate dehydrogenase complex.

We describe an Arabidopsis thaliana gene, ptlpd2, which codes for a protein with high amino acid similarity to lipoamide dehydrogenases (LPDs) from diverse species. Ptlpd2 codes for a precursor protein possessing an N-terminal extension predicted to be a plastid-targeting signal. Expression of the ptlpd2 cDNA in Escherichia coli showed the encoded protein possessed the predicted LPD activity. PTLPD2 protein, synthesized in vitro, was efficiently imported into isolated chloroplasts of Pisum sativum and shown to be located in the stroma. In addition, fusion proteins containing the predicted transit peptide of PTLPD2 or the entire protein fused at the N-terminus with the green fluorescent protein (GFP), showed accumulation in vivo in chloroplasts but not in mitochondria of A. thaliana. Expression of ptlpd2 was investigated by introducing ptlpd2 promoter-beta-glucuronidase (GUS) gene fusions into Nicotiana tabacum. GUS expression was observed in seeds, flowers, root tips and young leaves. GUS activity was highest in mature seeds, decreased on germination and increased again in young leaves. Expression was also found to be temporally regulated in pollen grains where it was highest in mature grains at dehiscence. Database searches on ptlpd2 sequences identified a second A. thaliana gene encoding a putative plastidial LPD and two genes encoding proteins with high similarity to the mitochondrial LPD of P. sativum.

Many parallel losses of infA from chloroplast DNA during angiosperm evolution with multiple independent transfers to the nucleus.

We used DNA sequencing and gel blot surveys to assess the integrity of the chloroplast gene infA, which codes for translation initiation factor 1, in >300 diverse angiosperms. Whereas most angiosperms appear to contain an intact chloroplast infA gene, the gene has repeatedly become defunct in approximately 24 separate lineages of angiosperms, including almost all rosid species. In four species in which chloroplast infA is defunct, transferred and expressed copies of the gene were found in the nucleus, complete with putative chloroplast transit peptide sequences. The transit peptide sequences of the nuclear infA genes from soybean and Arabidopsis were shown to be functional by their ability to target green fluorescent protein to chloroplasts in vivo. Phylogenetic analysis of infA sequences and assessment of transit peptide homology indicate that the four nuclear infA genes are probably derived from four independent gene transfers from chloroplast to nuclear DNA during angiosperm evolution. Considering this and the many separate losses of infA from chloroplast DNA, the gene has probably been transferred many more times, making infA by far the most mobile chloroplast gene known in plants.

Targeted inactivation of the plastid ndhB gene in tobacco results in an enhanced sensitivity of photosynthesis to moderate stomatal closure.

The ndh genes encoding for the subunits of NAD(P)H dehydrogenase complex represent the largest family of plastid genes without a clearly defined function. Tobacco (Nicotiana tabacum) plastid transformants were produced in which the ndhB gene was inactivated by replacing it with a mutant version possessing translational stops in the coding region. Western-blot analysis indicated that no functional NAD(P)H dehydrogenase complex can be assembled in the plastid transformants. Chlorophyll fluorescence measurements showed that dark reduction of the plastoquinone pool by stromal reductants was impaired in ndhB-inactivated plants. Both the phenotype and photosynthetic performance of the plastid transformants was completely normal under favorable conditions. However, an enhanced growth retardation of ndhB-inactivated plants was revealed under humidity stress conditions causing a moderate decline in photosynthesis via stomatal closure. This distinctive phenotype was mimicked under normal humidity by spraying plants with abscisic acid. Measurements of CO(2) fixation demonstrated an enhanced decline in photosynthesis in the mutant plants under humidity stress, which could be restored to wild-type levels by elevating the external CO(2) concentration. These results suggest that the plastid NAD(P)H:plastoquinone oxidoreductase in tobacco performs a significant physiological role by facilitating photosynthesis at moderate CO(2) limitation.

Acquisition of multiple virulence/avirulence determinants by potato virus X (PVX) has occurred through convergent evolution rather than through recombination.

Resistance to potato virus X (PVX) is determined by the product of a host resistance gene and a viral determinant specifying either virulence (resistance-breaking ability) or avirulence (resistance sensitivity). The viral coat protein is the determinant of resistance mediated by the host Nx gene while the 25 kDa movement protein is the determinant of Nb-mediated resistance. Group 1 and group 4 strains of PVX are avirulent or virulent respectively for both these determinants while group 2 and group 3 strains are virulent for one but avirulent for the other determinant. There are two alternative evolutionary mechanisms by which the various strain groups might have evolved: either by recombination between strains carrying virulence (or avirulence) determinants that evolved once only, or alternatively, by independent evolution of at least one virulence (or avirulence) determinant in distinct phylogenetic branches. These alternative hypotheses were investigated by (i) determining the complete genomic sequence of a group 1 and a group 4 strain and (ii) comparing the completely sequenced genomes of six isolates representative of the four strain groups. The analysis revealed the same phylogeny for all five PVX genes. Thus, there is no evidence that the PVX strain groups evolved by recombination.

Homeologous plastid DNA transformation in tobacco is mediated by multiple recombination events.

Efficient plastid transformation has been achieved in Nicotiana tabacum using cloned plastid DNA of Solanum nigrum carrying mutations conferring spectinomycin and streptomycin resistance. The use of the incompletely homologous (homeologous) Solanum plastid DNA as donor resulted in a Nicotiana plastid transformation frequency comparable with that of other experiments where completely homologous plastid DNA was introduced. Physical mapping and nucleotide sequence analysis of the targeted plastid DNA region in the transformants demonstrated efficient site-specific integration of the 7.8-kb Solanum plastid DNA and the exclusion of the vector DNA. The integration of the cloned Solanum plastid DNA into the Nicotiana plastid genome involved multiple recombination events as revealed by the presence of discontinuous tracts of Solanum-specific sequences that were interspersed between Nicotiana-specific markers. Marked position effects resulted in very frequent cointegration of the nonselected peripheral donor markers located adjacent to the vector DNA. Data presented here on the efficiency and features of homeologous plastid DNA recombination are consistent with the existence of an active RecA-mediated, but a diminished mismatch, recombination/repair system in higher-plant plastids.

An Arabidopsis gene encoding a chloroplast-targeted beta-amylase.

beta-Amylase is one of the most abundant starch degrading activities found in leaves and other plant organs. Despite its abundance, most if not all of this activity has been reported to be extrachloroplastic and for this reason, it has been assumed that beta-amylases are not involved in the metabolism of chloroplast-localized transitory leaf starch. However, we have identified a novel beta-amylase gene, designated ct-Bmy, which is located on chromosome IV of Arabidopsis thaliana. Ct-Bmy encodes a precursor protein which contains a typical N-terminal chloroplast import signal and is highly similar at the amino acid level to extrachloroplastic beta-amylases of higher plants. Expression of the ct-Bmy cDNA in E. coli confirmed that the encoded protein possesses beta-amylase activity. CT-BMY protein, synthesized in vitro, was efficiently imported by isolated pea chloroplasts and shown to be located in the stroma. In addition, fusions between the predicted CT-BMY transit peptide and jellyfish green fluorescent protein (GFP) or the entire CT-BMY protein and GFP showed accumulation in vivo in chloroplasts of Arabidopsis. Expression of the GUS gene fused to ct-Bmy promoter sequences was investigated in transgenic tobacco plants. GUS activity was most strongly expressed in the palisade cell layer in the leaf blade and in chlorenchyma cells associated with the vascular strands in petioles and stems. Histochemical staining of whole seedlings showed that GUS activity was largely confined to the cotyledons during the first 2 weeks of growth and appeared in the first true leaves at approximately 4 weeks.

Concurrent suppression of virus replication and rescue of movement-defective virus in transgenic plants expressing the coat protein of potato virus X.

A line of transgenic tobacco expressing the coat protein (CP) of potato virus X (PVX) was resistant against a broad spectrum of PVX strains. Inoculation of leaves and protoplasts with PVX expressing the jellyfish green fluorescent protein reporter gene revealed that this resistance mechanism suppressed PVX replication in the initially infected cell and systemic spread of the virus. Cell-to-cell movement was also slower in the resistant plants. The resistance at the level of replication was effective against wild-type PVX and also against movement-defective isolates with a frameshift mutation or deletion in the CP ORF. However, the cell-to-cell movement defect of the mutant viruses was rescued on the resistant plants. Based on these results it is proposed that the primary resistance mechanism is at the level of replication.

Cytoplasmic male sterility (CMS) in Lolium perenne L. 2. The mitochondrial genome of a CMS line is rearranged and contains a chimaeric atp 9 gene.

The most striking difference between the mtDNAs of the fertile L. perenne line LPSB21 and the male-sterile line CMS9B290, is the presence in the former and the absence in the latter of a 5.6-kb HindIII fragment. This difference between fertile and sterile lines was the starting point for a detailed molecular analysis of the mitochondrial genome in the region spanning the 5.6-kb HindIII fragment in fertile L. perenne and the corresponding region in CMS9B290. Restriction mapping and Southern-blot analyses indicated that rearrangement of the mitochondrial genome consistent with a deletion/insertion event had occurred in the sterile line. Nucleotide-sequence analysis of the rearranged region in CMS9B290 revealed the presence of (1) a novel chimaeric gene, orf-C9, comprising the first six codons of atp9 fused to a further 118 codons of an unknown sequence and (2) a truncated version of an open reading frame, orf-L, originally identified in LPSB21 mtDNA. Northern-blot analysis confirmed the absence of orf-L transcripts and the presence of orf-C9 transcripts in the mtRNA of CMS9B290.

Mutations conferring lincomycin, spectinomycin, and streptomycin resistance in Solanum nigrum are located in three different chloroplast genes.

A number of Solanum nigrum mutants resistant to the antibiotics spectinomycin, streptomycin and lincomycin have been isolated from regenerating leaf strips after mutagenesis with nitroso-methylurea. Selection of streptomycin- and spectinomycin-resistant mutants has been described earlier. Lincomycin-resistant mutants show resistance to higher levels of the antibiotic than used in the initial selection, and in the most resistant mutant (L17A1) maternal inheritance of the trait was demonstrated. The lincomycin-resistant mutant L17A1 and a streptomycin plus spectinomycin resistant double mutant (StSp1) were chosen for detailed molecular characterisation. Regions of the plastid DNA, within the genes encoding 16S and 23S rRNA and rps12 (3') were sequenced. For spectinomycin and lincomycin resistance, base changes identical to those in similar Nicotiana mutants were identified. Streptomycin resistance is associated with an A-->C change at codon 87 of rps12 (converting a lysine into a glutamine), three codons upstream from a mutation earlier reported for Nicotiana. This site has not previously been implicated in streptomycin resistance mutations of higher plants, but has been found in Escherichia coli. The value of these mutants for studies on plastid genetics is discussed.

Paternal inheritance of mitochondria and chloroplasts in Festuca pratensis-Lolium perenne intergeneric hybrids.

Organelle inheritance in intergeneric hybrids of Festuca pratensis and Lolium perenne was investigated by restriction enzyme and Southern blot analyses of chloroplast DNA (cpDNA) and mitochondrial DNA (mtDNA). All F1 hybrids exhibited maternal inheritance of both cpDNA and mtDNA. However, examination of backcross hybrids, obtained by backcrossing the intergeneric F1 hybrids to L. Perenne, indicated that both uniparental maternal organelle inheritance and uniparental paternal organelle inheritance can occur in different backcross hybrids.

A feature of the coat protein of potato virus X affects both induced virus resistance in potato and viral fitness.

The coat protein of PVX determines whether isolates of PVX are affected by Rx-mediated resistance in potato. Isolates with the coat protein of PVXHB are not affected by the resistance; those with the coat protein of PVXUK3 elicit an extreme resistance in the Rx potato that prevents virus accumulation, even on the inoculated leaf. In this paper we describe the analysis of a series of hybrid and mutant isolates of PVXHB and PVXCP4 which were inoculated to plants and protoplasts of Rx and rx cultivars of potato. From the virulence phenotypes of these isolates we conclude that elicitation of the resistance is affected by amino acids 121 and 127 of the viral coat protein, with codon 121 being the major determinant. PVXHB and hybrid or mutant isolates with lysine and arginine at positions 121 and 127 were able to overcome the resistance of Rx, whereas those with threonine and arginine were resistance sensitive both on plants and in protoplasts. The viral isolates with single mutations at either codon 121 or 127 were less infectious than the wild-type or double mutant isolates although, in protoplasts of the susceptible cultivar of potato, they accumulated as well as the wild-type virus. Taken together these data suggest that amino acids 121 and 127 affect a feature of the viral coat protein which may interact with cellular components involved in the spread of PVX and with the product of the Rx resistance gene.

A Potato Virus X Resistance Gene Mediates an Induced, Nonspecific Resistance in Protoplasts.

The Rx locus in potato controls extreme resistance to most isolates of potato virus X (PVX). The resistance is expressed in whole plants and in protoplasts. Rx-mediated resistance in protoplasts causes reduced accumulation of all PVX RNA species, including the (-) strand RNA after a lag of 8 hr postinoculation. In work reported elsewhere, we have shown that the Rx-breaking property of PVXHB was associated with the coat protein gene of PVXUK3 and PVXCP4. Here, we describe how a frameshift mutation in the coat protein gene had no effect on Rx resistance breaking but compromised the Rx-mediated resistance to PVXCP4. We also describe how in coinoculation experiments, the Rx-mediated resistance could be induced to affect PVXHB or cucumber mosaic virus (CMV). In these experiments, PVXHB or CMV was coinoculated to protoplasts (Rx genotype) together with an isolate of PVX, which is affected by Rx. We interpret this data to indicate that Rx-mediated resistance is induced when the PVX coat protein is produced in the infected cells and that the induced resistance mechanism is effective against viruses unrelated to PVX.

Cytoplasmic male sterility (CMS) in Lolium perenne L.: 1. Development of a diagnostic probe for the male-sterile cytoplasm.

Analysis of reciprocal crosses between nonrestoring fertile genotypes and restored male-sterile genotypes of Lolium perenne confirmed the cytoplasmic nature of the sterility trait. This prompted a search for a molecular probe that could be used to distinguish between fertile and cytoplasmic male-sterile (CMS) cytoplasms. We describe the identification and cloning of a 4.5-kb BamHI-HindIII restriction fragment from the mtDNA of the CMS line. The cloned fragment (pCMS45) failed to hybridise to sequences in the mtDNA of fertile lines and was thus capable of unambiguously distinguishing between fertile and CMS cytoplasms. The use of pCMS45 as a diagnostic probe provided a simple test for positive identification of young non-flowering plants carrying the CMS cytoplasm and also permitted confirmation at the molecular level of the maternal transmission of the CMS trait suggested by the genetic data.

Expression of photosynthesis-related gene fusions is restricted by cell type in transgenic plants and in transfected protoplasts.

We have analyzed the expression of chimeric genes in populations of protoplasts isolated from the photosynthetic and nonphotosynthetic tissues within leaves of transgenic tobacco plants and separated by fluorescence-activated cell sorting. Expression of transcriptional gene fusions controlled by promoters from photosynthesis-associated genes showed a striking dependence on cell type. These patterns of expression were preserved when the gene fusions were transfected into normal (nontransgenic) tobacco leaf protoplasts.

Targeting a foreign protein to chloroplasts using fusions to the transit peptide of a chlorophyll a/b protein.

We have constructed chimaeric genes consisting of sequences encoding the transit peptide and 4, 16, 24, 53 or 126 amino-terminal residues of the mature chlorophyll a/b binding (Cab) apoprotein fused to the Escherichia coli gene encoding beta-glucuronidase (GUS). These genes were introduced into tobacco plants and the fate of the fusion proteins they encode was analysed. Less than 1% of the total activity of fusion proteins containing the transit peptide and 4 (FP4) or 16 (FP16) amino-terminal amino acids of the mature Cab protein was associated with chloroplasts. Moreover, FP4 appears to be unprocessed. This is in striking contrast to fusion proteins containing the transit peptide and 24 (FP24), 53 (FP53) or 126 (FP126) amino-terminal residues of the mature Cab polypeptide. Approximately 98%, 96% or 75%, respectively, of the total activity of these fusion proteins was associated with purified intact chloroplasts, and protease protection experiments showed that of this, approximately 98%, 87% or 50%, respectively, was located within this organelle. Furthermore, both FP24 and FP53 appear to be processed. However, less than 10% of the activity of those fusion proteins translocated into chloroplasts was associated with thylakoid membranes.

Structure of melon rDNA and nucleotide sequence of the 17-25S spacer region.

Restriction enzyme and hybridization analysis of melon nuclear DNA suggests a homogenous rDNA population with a repeat unit of 10.2 kb. Several full length Hind III rDNA repeat units were cloned and one of these is described in detail. The regions coding for 25S, 17S and 5.8S rRNAs were located by crossed-contact hybridization and R-loop mapping. Introns were not observed. The nucleotide sequence of the internal transcribed spacer and flanking regions was determined and compared with the corresponding region from rice rDNA by dot matrix analysis. In addition, the extent of gross sequence homology between cloned melon and pea rDNA units was determined by heteroduplex mapping.