The use of the phosphomannose isomerase gene as alternative selectable marker for Agrobacterium-mediated transformation of flax (Linum usitatissimum)

In order to meet the future requirement of using non-antibiotic resistance genes for the production of transgenic plants, we have adapted the selectable marker system PMI/mannose to be used in Agrobacterium-mediated transformation of flax (Linum usitatissimum L.) cv. Barbara. The Escherichia coli pmi gene encodes a phosphomannose isomerase (E.C. 5.1.3.8) that converts mannose-6-phosphate, an inhibitor of glycolysis, into fructose-6-phosphate (glycolysis intermediate). Its expression in transformed cells allows them to grow on mannose-selective medium. The Agrobacterium tumefaciens strain GV3101 (pGV2260) harbouring the binary vector pNOV2819 that carries the pmi gene under the control of the Cestrum yellow leaf curling virus constitutive promoter was used for transformation experiments. Transgenic flax plants able to root on mannose-containing medium were obtained from hypocotyl-derived calli that had been selected on a combination of 20 g L⁻¹ sucrose and 10 g L⁻¹ mannose. Their transgenic state was confirmed by PCR and Southern blotting. Transgene expression was detected by RT-PCR in leaves, stems and roots of in vitro grown primary transformants. The mean transformation efficiency of 3.6%, that reached 6.4% in one experiment was comparable to that obtained when using the nptII selectable marker on the same cultivar. The ability of T1 seeds to germinate on mannose-containing medium confirmed the Mendelian inheritance of the pmi gene in the progeny of primary transformants. These results indicate that the PMI/mannose selection system can be successfully used for the recovery of flax transgenic plants under safe conditions for human health and the environment.     

Growth of Glomus intraradices and its effect on linseed (Linum usitatissimum L.) in hydroponic culture

This paper presents a hydroponic system for culturing and maintaining the VAM fungus Glomus intraradices in symbiosis with linseed (Linum usitatissimum L.) under greenhouse conditions in pure nutrient solution. It was possible to obtain large quantities of mycorrhizal host plant roots as well as extramatrical mycelium and chlamydospores free of impeding residues of solid substrate components. Starting from linseed donor plants inoculated in sand and transferred to the nutrient solution, new infections arose within the fast growing root system, hyphae spread out into the liquid and infected mycorrhiza-free receptor plants. Data for infection rates and plant growth parameters are presented. In comparsion to other culture systems for VAM fungi, the advantages of this hydroponic system are discussed and potential uses suggested.     

Dimorphic exine sculpturing in three distylous species ofLinum (Linaceae)

DistylousLinum grandiflorum, L. mucronatum, andL. pubescens have dimorphic pollen grains. In short-styled flowers the exine has monomorphic processes each with a ring of papillae. In long-styled flowers the exine has two types of processes: small processes terminating in a spinule and larger processes with a central spinule surrounded by a ring of spinuels, papillae or buttresses. The distylousLinum species of four different sections, in which morph-specific differences in the wall structure of stigmatic papillae have been reported, also exhibit exine dimorphism. The patterns of these dimorphisms are similar in all four sections in which distyly is present.     

Botanical analysis of a bundle of flax (Linum usitatissimum L.) from an early medieval site in northern Poland; a contribution to the history of flax cultivation and its field weeds

A bundle of flax (Linum usitatissimum L.) radiocarbon dated to 1210±70 uncal B.P. (830±90 cal A.D.) was analysed for its macrofossil content. Apart from stems, capsules and seeds of flax., a large number of diaspores (fruits and seeds) from other plants was identified. Field weeds were the most numerous taxa present, among them three flax field weeds,Spergula maxima, Camelina alyssum andCuscuta cpilinum. Development of the specific flax weed community is discussed. Indicator values are used to characterize the edaphic conditions of this early medieval flax field. The field weeds spectrum also suggests that this flax was sown as a summer crop after an earlier crop of millet.     

Cytogenetic analysis in Polypterus ornatipinnis (Actinopterygii, Cladistia, Polypteridae) and 5S rDNA

Polypteridae is a family of archaic freshwater African fish that constitute an interesting subject for the study of the karyological evolution in vertebrates, on account of their primitive morphological characters and peculiar relationships with lower Osteichthyans. In this paper, a cytogenetic analysis on twenty specimens of both sexes of Polypterus ornatipinnis the ornate "bichir", coming from the Congo River basin, was performed by using both classical and molecular techniques. The karyotypic formula (2n=36; FN=72) was composed of 26 M+10 SM. The Alu I banding, performed to characterize heterochromatin in this species, was mainly centromeric. Both the chromosome location of the ribosomal 5S and 18S rRNA genes were examined by using Ag-NOR, classical C-banding, CMA(3) staining and FISH. CMA(3) marked all centromerical regions and showed the presence of two GC rich regions on the p arm of the chromosome pair n°1 and on the q arm of the pair n°14. Staining with Ag-NOR marked the only telomeric region of the chromosome n°1 p arm. After PCR, the 5S rDNA in this species was cloned, sequenced and analyzed. In the 665bp 5S rDNA sequence of P.ornatipinnis, a conserved 120bp gene region for the 5S rDNA was identified, followed by a non-transcribed variable spacer (NTS) which included simple repeats, microsatellites and a fragment of a non-LTR retrotransposon R-TEX. FISH with 5S rDNA marked the subtelomeric region of the q arm of the chromosome pair n°14, previously marked by CMA(3). FISH with 18S rDNA marked the telomeric region of the p arm of the pair n°1, previously marked both by Ag-NOR and CMA(3). The (GATA)(7) repeats marked the telomeric regions of all chromosome pairs, with the exclusion of the n°1, n°3 and n°14; hybridization with telomeric probes (TTAGGG)(n) showed signals at the end of all chromosomes. Karyotype evolution in Polypterus genus was finally discussed, including the new data obtained.     

A rust-inducible gene from flax (fis1) is involved in proline catabolism

A gene fis1 from flax (Linum usitatissimum), which is induced in mesophyll cells at the site of rust (Melampsora lini) infection, is also expressed in vascular tissue, particularly in floral structures of healthy plants. This paper reports that the promoter controlling this expression is contained within 282 bp 5′ to the coding region and that fis1 gene induction is specifically by the rust pathogen and not by other fungal pathogens or by wounding. The fis1 gene has 73% homology with an Arabidopsis gene which encodes delta-1-pyrroline-5-carboxylate dehydrogenase (P5CDH) which is a part of the proline degradation pathway. Transgenic flax plants that either over-express fis1 or show reduced fis1 expression due to RNA-mediated gene silencing have an unaltered morphology. However, plants with reduced fis1 expression have markedly increased sensitivity to exogenous proline and show alteration in epidermal cell morphology, callose deposition and the production of hydrogen peroxide during proline-induced death. These lines, which show a biologically significant level of fis1 suppression, have an unaltered reaction to either virulent or avirulent rust infections, as do fis1 over-expression lines. These data indicate that the fis1 gene plays a role in proline metabolism and most likely encodes for a P5CDH enzyme. However, the precise role of fis1 and P5C catabolism in the development of rust disease remains unclear.     

Distyly and pollen dimorphism inLinum suffruticosum (Linaceae)

The Mediterranean distylousLinum suffruticosum has dimorphic pollen. That from short-styled plants has sub-monomorphic surface excrescences with minute papillae; that from long-styled plants has strongly dimorphic excrescences with conspicuous papillae. The differences are much like those described in species of other sections of the genus. Pollen of the related homostylousL. tenuifolium has monomorphic pollen. It combines characteristics of bothL. suffruticosum types but resembles that of the long-styled plants somewhat more closely. Ribbonlike styles of long-styled plants ofL. suffruticosum provide another feature which seems to be controlled by the S supergene thought to be associated with distylic incompatibility. The pollen ofL. suffruticosum andL. tenuifolium is very similar to that of some species of sectionLinastrum and would support their placement in that section.     

Localization of 5S rRNA Loci in Three Coregonid Species (Salmonidae)

In the present study the chromosome distribution of the 5S rDNA loci and its relation to the major rDNA genes were investigated in three Coregonid species (Salmonidae): Coregonus lavaretus, Coregonus peled and Coregonus albula, a family which has experienced large karyotype rearrangements along its evolution starting from a tetraploid ancestor. 5S PRINS/CMA₃ sequential staining together with previous data enabled us to locate 5S rRNA genes and nucleolar organizer regions (NORs) in the three species analyzed. PRINS revealed the 5S rDNA cluster at the distal part of the long arm of a similar submetacentric chromosome pair in the three species. Our data indicate that 5S rDNA clusters have probably conserved chromosomal location in the genus Coregonus, whereas 45S rDNA (NOR) sites are clearly differentiated, from a single locus in C. peled, to multiple loci in C. lavaretus and highly polymorphic multichromosomal location in C. albula.     

An unlinked 5 S ribosomal RNA gene in the archaebacterium, Thermococcus celer

Summary We have determined the nucleotide sequence of an unlinked 5 S rRNA gene region from a thermophilic archaebacterium, Thermococcus celer. This 5 S rRNA gene is flanked by a single tRNA^Asp sequence and appears to be transcribed as part of a very short operon consisting of only two gene sequences. Comparative studies indicate features in the 5 and 3 flanking sequences, which bear similarity with promoter and termination signals in eubacteria, but also reflect unusual features found in at least some archaebacteria. The evolution of this unlinked operon and the unusual features are discussed.     

The evolutionary breakdown of distyly inLinum tenuifolium (Linaceae)

Distyly inLinum tenuifolium L. is associated with a high degree of self-incompatibility. Breakdown in this system has occurred without the morphological rearrangement expected as the result of cross-over within the distyly supergene. Pollen-flow in both distylous and monomorphic populations is leptokurtic. A high proportion of intra-flower pollination occurs. Pollen production per ovule is reduced in the self-compatible race. Mean seed-set is increased. Individuals of the self-compatible race produce less vegetative growth and require a shorter time to reach reproductive maturity under cultivation. Increased fecundity appears to provide the selective advantage promoting the breakdown of self-incompatibility in this species. This is achieved through a massive shift of resources toward female reproductive function in plants of the monomorphic race. Subsequent colonization by this race has led to its increased distribution in C. & S. Europe.     

The 5S DNA units ofAcacia species (Mimosaceae)

The DNA sequence structure of 5S DNA units inAcacia species, including representatives from the three subgenera ofAcacia, have been determined. The data was interpreted to suggest that at least three lineages of 5S DNA sequences exist inAcacia and the proposal was made that the lineages be named5S Dna-1, 5S Dna-2, and5S Dna-3. The5S Dna-1 lineage was represented by units fromA. boliviana andA. bidwilli, the5S Dna-2 lineage by units fromA. melanoxylon, A. pycnantha, A. ulicifolia, A. boliviana, A. bidwillii, andA. albida, and the5S Dna-3 lineage by units fromA. bidwillii, A. boliviana, andA. senegal. Based on this interpretation of the sequence data, the Australian species of subg.Phyllodineae grouped together as a cluster, quite separate from the subgeneraAculeiferum andAcacia. As expected from the analyses of morphological characters, the 5S DNA units fromAcacia albida (syn.Faidherbia albida) were quite separate from the otherAcacia spp.     

Differential accumulation of monolignol-derived compounds in elicited flax (Linum usitatissimum) cell suspension cultures

Lignin and lignans share monolignols as common precursors and are both potentially involved in plant defence against pathogens. In this study, we investigated the effects of fungal elicitors on lignin and lignan metabolism in flax (Linum usitatissimum) cell suspensions. Cell suspension cultures of flax were treated with elicitor preparations made from mycelium extracts of Botrytis cinerea, Phoma exigua and Fusarium oxysporum F ssp lini. Elicitors induced a rapid stimulation of the monolignol pathway, as confirmed by the increase in PAL (phenylalanine ammonia-lyase, EC 4.1.3.5), CCR (cinnamoyl-CoA reductase EC 1.2.1.44) and CAD (cinnamyl alcohol dehydrogenase EC 1.1.1.195) gene expression and PAL activity. At the same time, CCR activity only increased significantly in F. oxysporum-treated cells 24 h post elicitation. On the other hand, CAD activity measured for coniferyl alcohol formation was transiently decreased but a substrate-specific activation of CAD activity was observed in F. oxysporum-treated cells when using sinapyl alcohol as substrate. The accumulation of monolignol-derived products varied according to the elicitor used. B. cinerea or P. exigua-elicited cell cultures were characterised by a reinforcement of the cell wall by a deposit of 8-O-4′-linked non-condensed lignin structures and phenolic monomers, while at the same time no stimulation of 8-8′-linked lignan or 8-5′-linked phenylcoumaran lignan accumulation was observed. Additionally, elicitation of cell cultures with F. oxysporum extracts even triggered a strong incorporation of monolignols in the non condensed labile ether-linked lignin fraction concomitantly with a decrease in lignan and phenylcoumaran lignan accumulation. Several hypotheses are proposed to explain the putative role of these compounds in the defence response of flax cells against pathogens. Electronic Supplementary Material Supplementary material is available for this article at and is accessible for authorized users. C. Hano and M. Addi contributed equally to this work.     

Mapping of the 18S and 5S ribosomal RNA genes in the fish Prochilodus argenteus Agassiz, 1829 (Characiformes,Prochilodontidae)

A single NOR-bearing chromosome pair was identified by silver nitrate staining in a previous study of the fish Prochilodus argenteus from the S ã o Francisco River (MG, Brazil), with a third metacentric chromosome sporadically bearing active NOR. The present study focused on an analysis of the chromosomal localization of both the major (45S) and the minor (5S) rRNA genes using FISH. The use of the 18S rDNA probe confirmed the previous Ag-NOR sites interstitially located in a large metacentric pair and also identified up to three other sites located in the telomeric regions of distinct chromosomes, characterizing an interindividual variation of these sites. In addition, the 5S rDNA site was revealed adjacent to the major NOR site, identified at the end of the large Ag-NOR bearing metacentric chromosome. In a few metaphases, an additional weak hybridization signal was observed in a third chromosome, possibly indicating the presence of another 5S rDNA cluster. Despite a lower karyotype diversification (2n=54 and FN=108) often observed among species of Prochilodontidae, variations involving both 45S and 5S rRNA genes could play an important role in their chromosome diversification.     

Non-concerted evolution of the RET76 satellite DNA family in Reticulitermes taxa (Insecta, Isoptera)

The evolutionary dynamics of satellite DNA is most often studied in canonical mating systems, where bisexuality and panmixis are the rule. In eusocial termites, the limited number of reproducers starting a new colony and the maintenance of the colony through few neotenics act as bottle-necks both in space and time. No data on repetitive DNA are available for Isoptera and for their peculiar reproductive strategy. Here we present the first satellite DNA family isolated in European Reticulitermes. RET76 is a G+C rich satellite embodying two sub-families with a 76 bp monomer. RET76 sequences are highly variable (sequence homology is lower than 80% within sub-families and lower than 68% in the entire family) and this variability is equally distributed among the eight analysed taxa, thus depicting a pattern of non-concerted evolution. The absence of variant fixation – together with the strict monomer length conservation – may be explained at the molecular level as due to functional constraints acting on these sequences, and/or at the organismic level by considering the involvement of eusociality in preventing or greatly reducing variant fixation, somehow mimicking an unisexual strategy.     

A tandemly repetitive centromeric DNA sequence of the fish Hoplias malabaricus (Characiformes: Erythrinidae) is derived from 5S rDNA

A substantial fraction of the eukaryotic genome consists of repetitive DNA sequences that include satellites, minisatellites, microsatellites, and transposable elements. Although extensively studied for the past three decades, the molecular forces that generate, propagate and maintain repetitive DNAs in the genomes are still discussed. To further understand the dynamics and the mechanisms of evolution of repetitive DNAs in vertebrate genome, we searched for repetitive sequences in the genome of the fish species Hoplias malabaricus. A satellite sequence, named 5SHindIII-DNA, which has a conspicuous similarity with 5S rRNA genes and spacers was identified. FISH experiments showed that the 5S rRNA bona fide gene repeats were clustered in the interstitial position of two chromosome pairs of H. malabaricus, while the satellite 5SHindIII-DNA sequences were clustered in the centromeric position in nine chromosome pairs of the species. The presence of the 5SHindIII-DNA sequences in the centromeres of several chromosomes indicates that this satellite family probably escaped from the selective pressure that maintains the structure and organization of the 5S rDNA repeats and become disperse into the genome. Although it is not feasible to explain how this sequence has been maintained in the centromeric regions, it is possible to hypothesize that it may be involved in some structural or functional role of the centromere organization.     

Analysis of LuPME3, a pectin methylesterase from Linum usitatissimum,revealed a variability in PME proteolytic maturation

Pectin methylesterase (PME) catalyzes the de-methylesterification of pectin in plant cell walls during cell elongation.¹ Pectins are mainly composed of α(1, 4)-D-galacturonosyl acid units that are synthesized in a methylesterified form in the Golgi apparatus to prevent any interaction with Ca²⁺ ions during their intracellular transport.² The highly methylesterified pectins are then secreted into the apoplasm³ and subsequently de-methylesterified in muro by PMEs. This can either induce the formation of pectin gels through the Ca²⁺ crosslinking of neighboring non-methylesterified chains or create substrates for pectin-degrading enzymes such as polygalacturonases and pectate lyases for the initiation of cell wall loosening.⁴ PMEs belong to a large multigene family. Sixty­six PME-related genes are predicted in the Arabidopsis genome.¹ Among them, we have recently shown that AtPME3 (At3g14310), a major basic PME isoform in A. thaliana, is ubiquitously expressed in vascular tissues and play a role in adventitious rooting.⁵ In flax (Linum usitatissimum), three genes encoding PMEs have been sequenced so far, including LuPME3, the ortholog of AtPME3. Analysis of the LuPME3 isoform brings new insights into the processing of these proteins.