A possible role of prolyl oligopeptidase during Linum usitatissimum (flax) seed development

Involvement of prolyl oligopeptidases (POPs) in the control of several mammalian peptide hormone signalling pathways has been studied extensively in recent years. POPs are ubiquitous enzymes, but little attention has been paid to understanding their function in plants. Using a cDNA-AFLP approach, two flax (Linum usitatissimum) POP ESTs were identified as being specifically expressed in the early stages of flax seed development. This specific expression was confirmed using real time RT-PCR and in situ hybridisation approaches. Seed expression of Arabidopsis POP genes was measured and showed no specificity. Comparison between results obtained with flax and Arabidopsis is discussed in order to address a hypothetic function for POPs during seed formation. These results provide the first insights into POP gene expression and hypothetical function in plants.     

Genetic control of polyunsaturated fatty acid biosynthesis in flax (Linum usitatissimum) seed oil

Summary The inheritance of two mutants of flax (Linum usitatissimum), having altered proportions of the C18 polyunsaturated fatty acids, linoleic and linolenic, was examined. Both lines, M1589 and M1722, are homozygous for a single gene mutation which reduces linolenic acid content from 34% to 22% and raises linoleic acid from 15% to 27%. Genetic analysis of crosses involving M1589, M1722 and their parental cultivar Glenelg revealed that these mutations are in different unlinked genes and exhibit additive (codominant) gene action. The symbolsLn1 andLn2 are proposed for the mutated genes in M1589 and M1722, respectively. Recombinant genotypes homozygous for the mutant alleles at both loci are very low in linolenic acid (2%) and high in linoleic acid (48%), with unaltered proportions of other fatty acids. The complete inverse correlation between linoleic and linolenic acids (r=-0.98) indicates that the mutations block the synthesis of linolenic acid at the linoleic desaturation step.     

RFLP and RAPD mapping in flax (Linum usitatissimum)

A map of flax (Linum usitatissimum) using restriction fragment length polymorphisms (RFLPs) and random amplified polymorphic DNAs (RAPDs), and comprising 15 linkage groups containing 94 markers, has been developed covering about 1000 cM. The mapping populations were the F₂ populations from two crosses between diverse cultivars. From one cross, CI1303 and Stormont Cirrus, 20 RFLP and 520 RAPD markers were analyzed. Thirteen RFLP and 80 RAPD markers were on the 15 linkage groups, in addition to one sequence-tagged site (STS). Seven polymorphic RAPD markers were found to have unusual segregation patterns. RAPDs were expressed as dominant markers, but for these markers a prevalence of the progeny lacked a band rather than the expected one-fourth ratio. However, these exceptions may be related to the instability of the genome of Stormont Cirrus in which stable and heritable genomic changes can be induced by environmental factors. The current map could be used for the identification of markers linked to loci controlling the ability to generate heritable changes in response to environmental growth conditions, and to develop anchor loci with STSs for a more general application. Received: 20 March 1999 / Accepted: 16 December 1999     

Characterization of transgenic sulfonylurea-resistant flax (Linum usitatissimum)

Summary Fourteen transgenic flax (Linum usitatissimum) lines, carrying a mutant Arabidopsis acetolactate synthase (ALS) gene selected for resistance to chlorsulfuron, were characterized for resistance to two sulfonylurea herbicides. Progeny of 10 of the 14 lines segregated in a ratio of 3 resistant to 1 susceptible, indicating a single insertion. Progeny of 1 line segregated in a 151 ratio, indicating two insertions of the ALS gene at independent loci. Progeny from 3 lines did not segregate in a Mendelian fashion and were likely the products of chimeric shoots. Resistance to chlorsulfuron was stably inherited in all lines. At the enzyme level, the transgenic lines were 2.5 to more than 60 times more resistant to chlorsulfuron than the parental lines. The transgenic lines were 25–260 times more resistant to chlorsulfuron than the parental lines in root growth experiments and demonstrated resistance when grown in soil treated with 20 g ha^-1 chlorsulfuron. The lines demonstrated less resistance to metsulfuron methyl; in root growth experiments, the transgenic lines were only 1.6–4.8 times more resistant to metsulfuron methyl than the parental lines. Resistance was demonstrated in the field at half (2.25 g ha^-1) and full (4.5 g ha^-1) rates of metsulfuron methyl.     

Variation within flax (Linum usitatissimum) and barley (Hordeum vulgare) in response to allelopathic chemicals

Summary A possible method of manipulating allelopathy would be to develop crop varieties showing an increased tolerance to allelopathic chemicals. We therefore examined four flax (Linum usitatissimum) varieties and two wild Linum species in the presence of p-coumaric acid and four barley (Hordeum vulgare) varieties in the presence of p-coumaric acid, scopoletin and wild oat (Avena fatua) extract. Analysis of variance indicates significant interaction between variety and treatment for shoot and root growth for seedling flax, shoot growth for older flax, and root growth for seedling barley. These differences in tolerance between varieties could be exploited to develop-varieties with greater tolerances to the allelochemicals produced by weeds or in crop residues and therefore potentially more tolerant of the presence of weeds.     

Heat-stress effects on reproduction and seed set in Linum usitatissimum L. (flax)

Heat stress can detrimentally affect the reproductive capacity of many plants. The effect of a 7 or 14 d heat stress on flowering, seed set, pollen viability and germinability of flax (Linum usistatissimum L.) was assessed under growth chamber conditions. An incremental (2 °C/h), cyclical (daytime high 40 °C and night‐time low 18 °C) heat stress was applied 12 d after the initiation of flowering. Although flower formation in flax was not affected by heat stress, boll formation and seed set were reduced with onset of the heat stress. On removal of heat stress the stressed plants showed a compensatory response, flowering and producing bolls at a greater rate than the control plants. Heat stress significantly prolonged flowering by 17 d. Boll weight and seed weight were reduced with heat stress and the number of malformed, sterile seed increased three‐fold after 14 d of heat stress. Pollen viability and appearance were negatively affected after 6 and 10 d of heat stress, respectively. Pollen germinability decreased by the sixth day of heat stress, with no pollen germinating by the tenth day. Effects of heat stress on pollen viability and germinability alone, which did not occur until after the sixth day of the stress, could not account for the decreased boll formation due to heat stress in flax. These observations suggest that a combined effect of heat stress on both pollen and ovules contributes to decreased boll formation and seed set in flax.     

Histological study of embryo-like structures initiated from hypocotyl segments of flax (Linum usitatissimum L.)

Cultivation of flax hypocotyl segments on MS medium supplemented with auxin (2,4-d, NAA) and combination of auxin (NAA) and cytokinin (BAP, zeatin) resulted in production of callus on the cut ends of segments and prolonged cultivation in globular structures resembling early stages of somatic embryos. Embryo-like structures protruded on the surface directly from the subepidermal layers of hypocotyl segments. Despite these globular structures closely resembling somatic embryos, histological observations did not reveal their embryogenic character–organogenesis was the predominant developmental morphogenic pathway. Based on our experiments, as well as on critical revision of existing reports on flax somatic embryogenesis, we conclude, that there has not yet been convincing histological proof of somatic embyogenesis from flax hypocotyl segments.     

Identification of microspore-derived plants in anther culture of flax (Linum usitatissimum L.) using molecular markers

The microspore origin of anther-culture-derived plants of flax was determined using inter-simple sequence repeat (ISSR) and randomly amplified polymorphic DNA (RAPD) markers. Polymorphic fragments between the two parents of the F₁ donor plants were identified and their segregation patterns in anther-culture-derived plants were used to elucidate the origin of those plants and to determine the degree of independence of plants regenerated from the same callus. Using one ISSR primer (UBC 889) and two RAPD primers (UBC 556 and 561), 12 out of 16 plants were unequivocally identified as being derived from microspores. Plants derived from the same callus had identical PCR patterns at five polymorphic loci and thus were likely derived from the same microspore. Therefore, it is proposed that the number of calli forming shoots be used to describe the anther culture efficiency in flax. Received: 3 February 1998 / Revision received: 8 June 1998 / Accepted: 8 July 1998     

SSR and morphological trait based population structure analysis of 130 diverse flax (Linum usitatissimum L.) accessions

A total of 130 flax accessions of diverse morphotypes and worldwide origin were assessed for genetic diversity and population structure using 11 morphological traits and microsatellite markers (15 gSSRs and 7 EST–SSRs). Analysis performed after classifying these accessions on the basis of plant height, branching pattern, seed size, Indian/foreign origin into six categories called sub-populations viz. fibre type exotic, fibre type indigenous, intermediate type exotic, intermediate type indigenous, linseed type exotic and linseed type indigenous. The study assessed different diversity indices, AMOVA, population structure and included a principal coordinate analysis based on different marker systems. The highest diversity was exhibited by gSSR markers (SI = 0.46; He = 0.31; P = 85.11). AMOVA based on all markers explained significant difference among fibre type, intermediate type and linseed type populations of flax. In terms of variation explained by different markers, EST-SSR markers (12%) better differentiated flax populations compared to morphological (9%) and gSSR (6%) markers at P = 0.01. The maximum Nei's unbiased genetic distance (D = 0.11) was observed between fibre type and linseed type exotic sub-populations based on EST-SSR markers. The combined structure analysis by using all markers grouped Indian fibre type accessions (63.4%) in a separate cluster along with the Indian intermediate type (48.7%), whereas Indian accessions (82.16%) of linseed type constituted an independent cluster. These findings were supported by the results of the principal coordinate analysis. Morphological markers employed in the study found complementary with microsatellite based markers in deciphering genetic diversity and population structure of the flax germplasm.     

Xylem changes in the correlative inhibition of lateral bud growth in flax (Linum usitatissimum L.)

Xylem or tracheary changes at the base of the cotyledonary buds of flax seedlings (Linum usitatissimum L.), released from inhibition by decapitation of the main apex were studied. The differentiation of xylem strands and/or tracheary elements was correlated with the growth in length of the lateral buds, especially 48–72 hr after the removal of the main apex. The xylem strands, connected to the hypocotylary stele or not, and the tracheary elements increased with age within and outside the strands of both non-decapitated and decapitated seedlings. In the latter, the differentiation of these structures, however, occurred much earlier and in greater abundance in the same regions. The early growth in length of lateral buds, 1 or 2 hr after decapitation, was correlated with the early development of tracheary perforations in the xylem strands. The xylary strands with perforated elements are known to be more efficient than those without them. Therefore, it is suggested that the inhibition of lateral-bud growth was due, in fact, to a lack of appropriate tracheary perforations in the bud xylem strands that were connected with the hypocotylary stele of flax seedlings.     

Variation in the isozymes of flax (Linum usitatissimum) genotrophs

Peroxidase, esterase, and acid phosphatase isozymes of environmentally induced L and S genotrophs, nuclear DNA reversion types, and the orginal plastic (Pl) type of the flax variety Stormont Cirrus have been compared by polyacrylamide gel electrophoresis. Differences were observed in particular line was not correlated with the nuclear DNA amount. The relationship between the isozyme pattern and the phenotypes of the lines in which they are expressed is discussed.     

Identification of quantitative trait loci contributing to Fusarium wilt resistance on an AFLP linkage map of flax (Linum usitatissimum)

 An AFLP genetic linkage map of flax (Linum usitatissimum) was used to identify two quantitative trait loci (QTLs) on independent linkage groups with a major effect on resistance to Fusarium wilt, a serious disease caused by the soil pathogen Fusarium oxysporum (lini). The linkage map was constructed using a mapping population from doubled-haploid (DH) lines. The DH lines were derived from the haploid component of F₂ haploid-diploid twin seed originating from a cross between a polyembryonic, low-linolenic-acid genotype (CRZY8/RA91) and the Australian cultivar ‘Glenelg’. The AFLP technique was employed to generate 213 marker loci covering approximately 1400 cM of the flax genome (n=15) with an average spacing of 10 cM and comprising 18 linkage groups. Sixty AFLP markers (28%) deviated significantly (P<0.05) from the expected segregation ratio. The map incorporated RFLP markers tightly linked to flax rust (Melamspora lini) resistance genes and markers detected by disease resistance gene-like sequences. The study illustrates the potential of the AFLP technique as a robust and rapid method to generate moderately saturated linkage maps, thereby allowing the molecular analysis of traits, such as resistance to Fusarium wilt, that show oligogenic patterns of inheritance. Received: 8 December 1997 / Accepted: 7 April 1998     

Another evidence for inhibitory effect of auxin in adventitious bud formation of decapitated flax (Linum usitatissimum L.) seedlings

Adventitious buds were formed on the hypocotyls of decapitated flax seedlings. Scanning electron and light microscopic examinations of hypocotyls showed that epidermal cells divided to produce meristematic spots from which several leaf primordia were formed. Between leaf primordia and the original vascular tissues of hypocotyls, new xylem cells were formed which connected them. About 10, 30 and 60% of adventitious buds were formed on upper, middle and basal parts of hypocotyls of decapitated seedlings, respectively. Removal of apical meristem together with longer hypocotyl zero to four cm long below the apical meristem) induced higher percentage of adventitious bud formation in the remaining hypocotyl. When the entire hypocotyl was cut into 16 segments (0.25 cm each) and these segments were cultured on MS medium containing 3% sucrose and 0.8% agar, adventitious buds were mainly formed in the lowest five segments. These results suggested that there was a gradient of inhibitory factor(s) from apical to basal part of hypocotyl with respect to adventitious bud formation. Auxin transport inhibitors, morphactin and TIBA induced adventitious bud formation on intact seedlings by suppressing the basipetal movement of auxin.     

Cloning and expression of (R)-hydroxynitrile lyase from Linum usitatissimum (flax)

The gene encoding for (R)-hydroxynitrile lyase ((R)-HNL) from Linum usitatissimum has been cloned by polymerase chain reaction using 3′,5′-RACE (rapid amplification of cDNA ends). The resulting clone contained an open reading frame of 1266 bp corresponding to a protein of 422 amino acids (45.8 kDa), which shows significant homologies to zinc-dependent formaldehyde dehydrogenases and alcohol dehydrogenases from various organisms. The dimeric active enzyme was expressed in Escherichia coli as N-terminal hexa-histidine fusion protein allowing the purification of homogeneous protein in one step. The formation of inclusion bodies could be reduced using a thioreductase deficient E. coli strain as a host and performing expression of (R)-HNL at 28°C. Under these conditions recombinant (R)-HNL was obtained with a specific activity of 76 U/mg.     

The biosynthesis of cyanogenic glucosides in Linum usitatissimum (linen flax) in Vitro

Microsomal preparations from dark-grown Linum usitatissimum (linen flax) seedlings synthesize acetone cyanohydrin, the precursor of the cyanogenic glucoside linamarin, from valine in the presence of NADPH. N-Hydroxyvaline and isobutyraldoxime, which are predicted intermediates in the pathway, are also converted into products. These microsomal preparations also convert isoleucine into 2-butanone cyanohydrin the precursor of lotaustralin. The biosynthetic activity is located exclusively in the developing cotyledons.     

Intraspecific 5S rRNA gene variation in flax,Linum usitatissimum (Linaceae)

A relatively large proportion of the flax genome (~ 3%) is comprised of 5S rRNA genes. This study focuses on the intraspecific sequence variation among five distinct groups of 5S rRNA genes. The results indicate that group 1 and 2 5S rRNA genes most closely resemble other angiosperm 5S genes, while groups 3–5 are highly divergent. Sequence variation is higher in the spacer region compared to the transcribed region for all pairwise comparisons. The large degree of sequence variation observed in this study is discussed with respect to genome organization and proposed models for repetitive sequence maintenance.     

Evidence of the domestication history of flax (Linum usitatissimum L.) from genetic diversity of the sad2 locus

A phylogenetic analysis was conducted on 34 alleles of 2.5 kb sized stearoyl-ACP desaturase II (sad2), obtained from 30 accessions of cultivated and pale flax (Linum spp.), to elucidate the history of flax domestication. The analysis supports a single domestication origin for extant cultivated flax. The phylogenetic evidence indicates that flax was first domesticated for oil, rather than fibre. The genetic diversity of the sad2 locus in cultivated flax is low when compared to that of the pale flax assayed. An absolute archaeological date could be applied to the synonymous substitution rate of sad2 in cultivated flax, yielding a high estimate of 1.60–1.71×10⁻⁷ substitutions/site/year. The occurrence of nonsynonymous substitutions at conserved positions of the third exon in alleles from cultivated flax suggests that the locus may have been subjected to an artificial selection pressure. The elevated synonymous substitution rate is also compatible with a population expansion of flax since domestication, followed by a population decline in historic times. These findings provide new insight into flax domestication and are significant for the continuous exploration of the flax germplasm for utilization.