Synthesis of Cell Wall Galactans from Flax (Linum usitatissimum L.) Suspension-Cultured Cells

Cell walls of flax suspension-cultured cells contain both ß1     

Evidence for Several Galactan Synthases in Flax (Linum usitassimum L.) Suspension-Cultured Cells

A membrane fraction from flax cells was able to incorporate[¹⁴C]galactose from UDP-D-[¹⁴C]galactose in vitro. The productsof the reaction, characterized by methylation analysis, consistedof a rß-1,4-galactan (solubilized mainly in water)and a rß-1,3- rß-1,6-galactan (solubilizedmainly in alkali). These results indicated the presence of severalgalactan synthase complexes, as did a profile of the relationshipbetween pH and activity which revealed both a maximum at pH6.5 and a shoulder at pH 8. Moreover, galactan synthase activitieswere found at two densities: 1.125 g cm^–3 (Golgi membranes)and 1.07–1.08 g cm^–3 (corresponding to low-densityvesicles). Partial characterization of one enzymatic system (maximaly activeat pH 8 in the presence of 5 mM MgCl₂) was achieved. The K_mfor UDP-galactose and V_max were 38 µM and 4.5 nmol h^–1(mg protein)^–1, respectively. (Received June 6, 1993; Accepted September 22, 1993)     

亚麻EST序列中SSR标记的筛选

利用亚麻NCBI数据库中的7 941条亚麻EST序列进行SSR的筛选,共发现222个SSR,占整个EST数据库的2.73%,其中三核苷酸重复单元的EST-SSR占总SSR的72.1%,二核苷酸和四核苷酸二者出现的频率基本相近,分别占总SSR的14.4%和13.5%.AGAA是四核苷酸中的优势重复类型,占四核苷酸重复类型的67.67%.设计的21对EST-SSR引物中有18对在10个亚麻材料中有扩增产物,占设计引物的85%,有14对产物条带比较清晰并具有多态性.基于SSR标记进行聚类分析,可将10个亚麻材料划分为3个组.本研究建立的亚麻SSR标记,为亚麻遗传多样性鉴定、分子作图等研究提供了一种有效的分子标记系统.     

Interaction of Genes Controlling Some Morphological Features of Flax (Linum usitatissimum L.)

Russian Journal of Genetics - The interaction of 11 genes of flax which control the color of the hypocotyl, flowers and seeds, as well as deformation of petals, was evaluated by genetic analysis....     

Immunochemical Characteristics of Isoperoxidases from Two Environmentally-induced Flax Genotrophs (Linum usitatissimum L.)

The homology between the acidic isoperoxidases from two environmentally-inducedflax genotrophs, L and S, was examined with antisera raisedagainst purified isozymes from S stem tissue. Peroxidases S1,S2 and S4 were found to be immunologically indistinguishablefrom their counterparts L1, L2 and L4 based on results fromimmunodiffusion, immunoinhibition and immunoprecipitation experiments.Corresponding isozymes from S and L, despite displaying differencesin apparent molecular weight, were shown to have identical plvalues. These results support our view that post-translationalmodification of the carbohydrate moiety of the isoperoxidasesfrom L and S is responsible for their differences on polyacrylamidegel electrophoresis. The affinity of the antisera toward threehorseradish peroxidases was also studied and the presence ofthree antigenically distinct groups of peroxidases in plantsis suggested. Key words: Flax peroxidase, horseradish peroxidase, isoperoxidases, homology     

Cyclopeptides of Linum usitatissimum.

Cyclolinopeptide A (CLA), a cyclic nonapeptide from linseed, possesses strong immunosuppressive and antimalarial activity along with the ability to inhibit cholate uptake into hepatocytes. The structure of the peptide was studied extensively in solution as well as in the solid state. It is postulated that both the Pro-Pro cis-amide bond and an 'edge-to-face' interaction between the aromatic rings of two adjacent Phe residues are important for biological activity. Structure-activity relationship studies of many linear and cyclic analogues of CLA suggest that the Pro-Xxx-Phe sequence and the flexibility of the peptide are important for the immunosuppressive activity.     

Identifying Novel Polymorphic Microsatellites from Cultivated Flax (Linum usitatissimum L.) Following Data Mining

One of the major concerns in genetic characterization and breeding of cultivated flax is the lack of informative microsatellite markers (SSRs). In this regard, the development of SSRs using molecular methods might be time-consuming, laborious, and expensive. On the other hand, using bioinformatics to mine sequences in public databases enables a cost-effective discovery of SSRs. A total of 3,242 Linum usitatissimum genomic sequences were surveyed for the identification of SSRs. Among them, 118 non-redundant sequences containing repeats were selected for designing primers. The most abundant motifs were tri- (72.4%) and dinudeotide (16.6%), within which AGG/CCT and AG/CT were predominant. Primers were tested for polymorphism in 60 L. usitatissimum cultivars/accessions including 57 linseed and three fiber flax. Eighty-eight pairs gave amplifications within the expected size range while 60 pairs were found to be polymorphic. The mean number of alleles amplified per primer was 3.0 (range, 2–8; 180 total alleles). The mean polymorphism information content (PIC) value was 0.39 (range, 0.06–0.87), and the highest average PIC was observed in dinucleotide SSRs (0.41). The SSR data mining presented here demonstrates the usefulness of in silico development of microsatellites. These novel genomic SSR markers could be used in genetic diversity studies, the development of genetic linkage maps, quantitative trait loci mapping, association mapping, and marker-assisted selection.     

不同品种亚麻籽粒中主要脂肪酸含量的变化

不同亚麻品种‘陇亚7号’、‘82（50）’、‘匈牙利3号’和‘范昵’籽粒中亚麻酸含量最高,其次是油酸、亚油酸和硬脂酸,棕榈酸含量最低。棕榈酸和亚油酸在籽粒发育成熟的初期增长速度较快,含量较高,但随着籽粒逐渐发育成熟而下降,硬脂酸也如此,后者下降幅度较小。油酸和亚麻酸含量随着籽粒发育成熟进程而渐增,油酸含量增加幅度较小。不同品种的籽粒完全成熟时,棕榈酸、硬脂酸、亚油酸和亚麻酸含量有异,同一基因型的亚麻籽粒完全成熟时,开花晚的油酸含量较低,而亚麻酸含量较高。     

Rm Differences in Leaf Malate Dehydrogenases of Flax (Linum usitatissimum) Genotrophs: Apparent Developmental Effects

Fieldes, M. A. and Gray, T. J. 1988. R_m differences in leafmalate dehydrogenases of flax (linum usitatissimum) genotrophs:apparent developmental effects.—J. exp. Bot. 39: 499–509. Malate dehydrogenase (MDH) isozyme relative mobility (R_m) wasexamined in leaf extracts of Durrant's large (L) and small (S)flax genotrophs. Within both L and S there were differencesin R_m between leaves sampled from different positions down themain stem and between leaves sampled from plants of differentages. For leaves sampled from plants which were at the onsetof flowering, the R_m differences from the apex to the base ofthe stem showed similar trends in L and S. However, the neteffect of the trend for L was a linear increase in R_m from apexto base, which did not occur in S. The changes in R_m which occurredin apical leaves as the plants aged were also different in Land S; R_mdecreased in L and increased in S during the growthperiod just prior to flowering. The possible relationship betweenthese differences in the changes in MDH R_m within L and S, previouslyreported differences in the changes in peroxidase (PER) isozymeR_m and the morphological/developmental differences between Land S is discussed. In addition, the experimentation demonstratedthat ‘negative’ bands detected in MDH-stained gelsunder certain staining conditions appear to correspond to PERisozymes and effectively mean that PER and MDH isozyme R_m'scan be obtained from the same electrophoretic gels. Key words: Malate dehydrogenase, peroxidase, relative mobility, flax     

Peroxidase Activity in Linum usitatissimum L

Peroxidase activity was measured at three stages, from seedlingto maturity, in leaf and stem tissue of two genotypes of Linumusitatissimum. The plants were grown throughout in growth chambers,allowing close control of the environmental conditions. Therewere large and consistent differences between activities inthe genotypes, between leaf and stem tissue, and between dialyzedand undialyzed extracts. Dialysis appeared to remove inhibitor(s).The consistency of the genotypic differences under these conditionspermitted reliable comparisons, from the seedling stage to maturity,to be made, a finding relevant to inheritance studies of peroxidaseactivity.     

Polymorphic microsatellite loci in Linum usitatissimum

We report the characterization of 28 polymorphic microsatellite markers in Linum usitatissimum that allow distinguishing almost all cultivars of both flax and linseed. Polymorphism was low, ranging from two to 10 alleles per locus in the 93 cultivars screened. Linkage disequilibrium was found at about a third of the pairs of loci likely due to self‐fertilization and strong selection by breeders. We tested these loci for cross‐amplification in nine additional species of Linum and found that three species amplified a majority of loci.     

Composition and Distribution of Cell Wall Phenolic Compounds in Flax (Linum usitatissimum L.) Stem Tissues

Cell wall phenolic compounds were analysed in xylem and bastfibre-rich peels of flax stems by biochemical, histochemicaland ultrastructural approaches. Localization of cell wall phenolicsby the enzyme-gold method using laccase revealed several goldparticle distribution patterns. One of the major types (an evendistribution of single gold particles) was present mainly inxylem, while the other (compact branched groups of ten–40gold particles) was found both in xylem and fibre cells. Thelignin content of the stem parts was estimated by the Klasonprocedure and by the thioglycolic acid assay, and the phenolicproducts recovered after alkaline cupric oxide oxidation ofcell walls were analysed by GC. By combining chemical analysisdata and the frequency of various gold particle types withinthe tissues, different patterns of gold particle distributioncould be ascribed to certain cell wall phenolics; lignin wasstained as evenly distributed single gold particles, while branchedclusters represented hydroxycinnamic acids. The Klason proceduredid not remove all the non-lignin components from flax fibres,known for their highly crystalline cellulose, and considerablyoverestimated the lignin content. The thioglycolic acid assayresults were consistent with GC and microscopic observations.Copyright 2000 Annals of Botany Company Linum usitatissimum L., bast fibres, cell wall, lignin, hydroxycinnamic acids     

Floral-Dip Transformation of Flax (Linum usitatissimum) to Generate Transgenic Progenies with a High Transformation Rate

Agrobacterium-mediated plant transformation via floral-dip is a widely used technique in the field of plant transformation and has been reported to be successful for many plant species. However, flax (Linum usitatissimum) transformation by floral-dip has not been reported. The goal of this protocol is to establish that Agrobacterium and the floral-dip method can be used to generate transgenic flax. We show that this technique is simple, inexpensive, efficient, and more importantly, gives a higher transformation rate than the current available methods of flax transformation.In summary, inflorescences of flax were dipped in a solution of Agrobacterium carrying a binary vector plasmid (T-DNA fragment plus the Linum Insertion Sequence, LIS-1) for 1 - 2 min. The plants were laid flat on their side for 24 hr. Then, plants were maintained under normal growth conditions until the next treatment. The process of dipping was repeated 2 - 3 times, with approximately 10 - 14 day intervals between dipping. The T1 seeds were collected and germinated on soil. After approximately two weeks, treated progenies were tested by direct PCR; 2 - 3 leaves were used per plant plus the appropriate T-DNA primers. Positive transformants were selected and grown to maturity. The transformation rate was unexpectedly high, with 50 - 60% of the seeds from treated plants being positive transformants. This is a higher transformation rate than those reported for Arabidopsis thaliana and other plant species, using floral-dip transformation. It is also the highest, which has been reported so far, for flax transformation using other methods for transformation.     

Peroxidase Activity in Linum usitatissimum L.

Peroxidase activity was measured at three stages, from seedlingto maturity, in stem tissue of two genotypes of Linum usitatissimum,and their reciprocal F₁ hybrids. The plants were grown throughoutin growth chambers, allowing close control over the environmentalconditions. There were large and consistent differences betweenthe activities of the parental genotypes, and between dialysedand undialysed extracts. Activities in both parents and theF₁ were expressed on a logarithmic scale. The activity of theF₁ fell, with one exception, between the activities of the twoparents. The relationship of F₁ activity to the mean of theparental activities fluctuated with the stage of growth.     

Peroxidase activity in Linum usitatissimum L.

Summary Crosses were made, in all combinations, between 2 parental genotypes of Linum and their reciprocal F ₁ hybrids. The parents and progeny obtained were grown in controlled environmental conditions and sampled at 35 and 70 days after germination to determine, on an individual plant basis, total plant fresh weight and peroxidase activity of main stem tissue. Peroxidase activity required transformation to a log₁₀ scale, whereas the original linear scale was satisfactory for plant weight. There was no correlation between plant weight and corresponding peroxidase activity. Pronounced heterosis appeared in the F ₁'s for both characters at sample 1, but this heterosis had declined at sample 2 and in the F ₂'s. Heterosis operated in a positive direction for plant weight and in a negative direction for peroxidase activity. No consistent differences were found amongst the variances of segregating or non-segregating generations for either character.     

Long-Term Phosphate Starvation and Respiratory Metabolism in Suspension-Cultured Catharanthus roseus Cells

In order to clarify the metabolic adaptation of respiratorypathways in plants to limited levels of P_i, the effects of long-termstarvation of P_i on the activities of various enzymes relatedto respiratory metabolism were examined in suspension-culturedCatharanthus roseus cells. When the activities were expressedas units per g fresh weight, only those of phosphoenolpyruvate-hydrolyzing(PEP-hydrolyzing) enzyme (which may possibly be equivalent tothe acid phosphatase activity derived from vacuoles) and PEPcarboxylase were higher in the Pⁱ-starved cells than in controlcells. Activities of other enzymes in the Pⁱ-starved cells werelower than or similar to those of the control cells. Time-coursestudies indicated that PEP-hydrolyzing activity was inducibleby starvation of Pⁱ. However, in contrast to the results reportedby Duff et al. [(1989a) Plant Physiol. 90: 1275.], fluctuationsin the activity of PP¹:fructose-6-phosphate 1-phosphotransferaseduring starvation of Pⁱ were similar to those in levels of phosphofructokinaseand 6-phosphogluconate dehydrogenase. These data suggest thatthe concept of the phosphate starvation-inducible ‘bypasses’,which are engineered via the coarse control (i.e., induction)of specified enzymes and were proposed initially by Duff etal. in Brassica nigra cells, is not directly applicable to Catharanthusroseus cells in suspension. Tracer experiments using [U-¹⁴C]glutamineindicated that a significant proportion of respiratory substratescould be supplied from the enlarged pool of amino acids duringstarvation of Pⁱ. These assumptions are supported by the observedfluctuations in levels of free amino acids and of protein inP¹-fed and P¹-deficient Catharanthus roseus cells. ¹Part 41 in the series ‘Metabolic Regulation in PlantCell Cultrue’ ²Present Address: Morinaga Mild Industry, 5-1-83, Higashihara,Zamma-shi, Kanagawa, 228 Japan     

Outlier Loci and Selection Signatures of Simple Sequence Repeats (SSRs) in Flax (Linum usitatissimum L.)

Genomic microsatellites (gSSRs) and expressed sequence tag-derived SSRs (EST-SSRs) have gained wide application for elucidating genetic diversity and population structure in plants. Both marker systems are assumed to be selectively neutral when making demographic inferences, but this assumption is rarely tested. In this study, three neutrality tests were assessed for identifying outlier loci among 150 SSRs (85 gSSRs and 65 EST-SSRs) that likely influence estimates of population structure in three differentiated flax sub-populations (F _ST?=?0.19). Moreover, the utility of gSSRs, EST-SSRs, and the combined sets of SSRs was also evaluated in assessing genetic diversity and population structure in flax. Six outlier loci were identified by at least two neutrality tests showing footprints of balancing selection. After removing the outlier loci, the STRUCTURE analysis and the dendrogram topology of EST-SSRs improved. Conversely, gSSRs and combined SSRs results did not change significantly, possibly as a consequence of the higher number of neutral loci assessed. Taken together, the genetic structure analyses established the superiority of gSSRs to determine the genetic relationships among flax accessions, although the combined SSRs produced the best results. Genetic diversity parameters did not differ statistically (P?>?0.05) between gSSRs and EST-SSRs, an observation partially explained by the similar number of repeat motifs. Our study provides new insights into the ability of gSSRs and EST-SSRs to measure genetic diversity and structure in flax and confirms the importance of testing for the occurrence of outlier loci to properly assess natural and breeding populations, particularly in studies considering only few loci.     

The ubiquitin-encoding multigene family of flax, Linum usitatissimum.

Ubiquitin (Ubq), a 76-amino acid (aa) protein, is found in all eukaryotic organisms and is one of the most conserved proteins so far studied. It is implicated in many cellular processes. The Ubq-encoding genes (ubq) are generally present as a multigene family. In flax, we have estimated that this multigene family contains at the most ten members. The initial flax ubq sequences were isolated from a flax genomic library in lambda EMBL4 using a heterologous Arabidopsis thaliana ubq probe. An 916-bp fragment from one of the phage clones was subcloned and sequenced. The aa sequence derived from the nucleotide sequence of this fragment is identical to that of other plant Ubqs. This fragment was then used to isolate additional flax ubq clones. In all, eleven phage lambda clones, which represent six members of the gene family, were restriction-mapped and characterized. These six members are represented as three monomers, three poly-Ubqs, one hexamer and two tetramers. They can be present at either a single locus (two of the monomers and one of the poly-Ubqs) or at two loci (the remaining three genes). The other four members of the family are yet to be cloned and characterized.