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.     

Influence of methyl jasmonate on podophyllotoxin and 6-methoxypodophyllotoxin accumulation in Linum album cell suspension cultures

The accumulation of podophyllotoxin (PTOX) and 6-methoxypodophyllotoxin (6MPTOX) was enhanced about twofold in the suspension culture of Linum album line 2-5 aH following the addition of methyl jasmonate (MeJas) to the cultivation medium, reaching 7.69±1.45 mg/g dry weight and 1.11±0.09 mg/g dry weight, respectively. There was no increase in 6MPTOX accumulation following the addition of MeJas to suspension cells of L. album line X4SF, whereas PTOX accumulation was enhanced about tenfold to 0.49±0.10 mg/g dry weight. Phenylalanine ammonia-lyase activity increased immediately after the addition of MeJas to a cell suspension culture of line X4SF, reaching a maximum between 4 h and 1 day after elicitation, while cinnamyl alcohol dehydrogenase activity and the lignin content of the cells were not affected.     

Alkaloid production in elicited cell suspension cultures of Erythrina americana Miller

A number of species of the genus Erythrina are rich in secondary metabolites, particularly phenolics and alkaloids that exhibit interesting anti-inflammatory, anti-plasmodial, bactericidal, curariform and fungicidal activities. Unfortunately, the isolation of these compounds through the extraction of organs and seeds of whole plants is becoming more difficult since the natural growing areas of many of the species, and particularly of Erythrina americana, are being urbanised. Plant tissue culture not only constitutes a viable method through which to preserve the species, but may also represent a constant and stable source of target alkaloids. Currently, however, in vitro systems, and especially cell suspension cultures, only accumulate low levels of the desired products. A number of strategies for increasing production have been proposed, the most successful of which involves elicitation of suspension cells with plant growth regulators. In this context, excellent results have been reported following elicitation of cell cultures derived from different species and genera with jasmonic acid and methyl jasmonate. The present paper provides a brief review of the novel approaches to the use of Erythrina alkaloids that have recently been described, and of the advances that have been made in the formation of tissue cultures of E. americana and their subsequent elicitation in attempts to augment alkaloid accumulation.     

Evaluation of putative seed-specific promoters for Linum usitatissimum

The GUS reporter gene was used to test four different putativeseed-specific promoters in developing and mature seeds, leaves and roots fromlinseed flax (Linum usitatissimum). The promoters testedincluded the regulatory regions of the -ketoacyl-CoA synthase gene (KCS)and the napin protein gene from Brassica napus, thepromoter regions of the 'unknown seed protein' (USP), and a legumin proteingene(LeB4) from Vicia faba and the CaMV 35S promoter (positivecontrol). The promoter-GUS constructs were inserted into L.usitatissimum via Agrobacterium mediatedtransformation, and GUS activity evaluated using histochemical andfluorimetrical assays. All the promoters showed some activity, but only CaMV35S, LeB4 and USP exhibited an expression level high enough to be useful inlinseed flax. Plants with USP-GUS showed the earliest GUS activity at 5 to 6days after flowering (daf) and persisting until 40 daf. Expression of GUS underthe control of the LeB4 promoter was measurable 11 daf and was still detectableat 40 daf. The KCS-GUS construct showed a low level of GUS activity between 14daf and 40 daf. Plants transformed with USP-GUS or LeB4-GUS exhibited a lowlevel of GUS activity in leaves and roots of some of the transformants,indicating the need for generating large numbers of primary transformants,followed by careful evaluation and selection for ones with not only the desiredlevel of expression, but also the desired spatial and temporal expression.     

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.     

Purification of several pectin methyltransferases from cell suspension cultures of flax (Linum usitatissimum L.)

Three pectin methyltransferases (PMT5, PMT7, PMT18; EC 2.1.1.6.x) were solubilized from the endo-membrane complex of flax cells, with 0.05% Triton X-100. After a 3 step-chromatography procedure, PMT7 and PMT5 were purified to apparent homogeneity. PMT5 and PMT7 differed regarding their optimum pH (5 or 7), the methyl acceptor (low or highly methylesterified pectin), their focusing pH range (6-7 or 8-9) and relative molecular mass (40 +/- 5 or 110 +/- 10 kDa). SDS-PAGE of PMT5 and PMT7 did not reveal bands at 40 or 110 kDa but only a silver stained band of about 18 kDa. Two independent methods (photo labelling and enzymatic activity) showed that this silverstained band corresponded to a methyltransferase with affinity for pectins. This polypeptide was of the same size as the enzyme designed PMT18 (18 +/- 3 kDa; pl 4-4.5) recovered during size exclusion chromatography of either PMT7 or PMT5, suggesting that PMT18 bears the catalytic site of PMT5 and PMT7.     

Defense responses elicited in pine cell suspension cultures

To understand mechanisms of disease resistance in pine trees, we took advantage of the fact that suspension cultured cells exhibit many of the defense responses that are characteristic of intact tissues. In this study, we measured constitutive and elicitor-induced levels of ethylene production, chitinase activity and glucanase activity in cells of loblolly pine (Pinus taeda L). Increased ethylene production was induced similarly by a live fungus (Ophiostoma minus Hedgc. H.P. Sydow) and chitosan, a general elicitor. Culture age, relative to the most recent transfer, affected the constitutive level of all defense responses. Culture age also had a pronounced effect on the ability of the cells to produce ethylene and cellular chitinase, but not on secreted chitinase, cellular glucanase, secreted glucanase, or lignification. In older cultures, elicitation induced a 4- to 10-fold increase in ethylene production and a 2-fold increase in cellular chitinase, secreted chitinase and cellular glucanase. Chitosan elicitation did not affect secreted glucanase. The overall regulation of the defense response in pine cells appears complex, but individual components of the response can be differentially induced in cell cultures under appropriate experimental conditions.     

Cellulase elicitor induced accumulation of capsidiol in Capsicum annumm L. suspension cultures

When growth-phase cell suspension cultures of Capsicum annuum were treated with cellulase-elicitor preparation at 3 μg/ml, the level of capsidiol was transiently increased in the culture media rather than in the cells reaching its maximum approx 24 h after treatment. With methyl jasmonate it took 18 h. Elicitor treatment doubled phospholiphase A₂ (PLA₂) activity but simultaneous treatment with aristolochic acid, a PLA₂ inhibitor, inhibited sesquiterpenoid accumulation as well as PLA₂ activity. Mastoparan, a G protein activator, treatment also increased PLA₂ activity and capsidiol production. Taken together, the present study shows that induction of capsidiol production in the C. annuum is mediated by PLA₂ activation.     

Relative mobility and activity of leaf malate dehydrogenase in flax(Linum usitatissimum) genotrophs and genotypes

Malate dehydrogenase (MDH) band relative mobility (R _m) and activity were examined in leaf extracts of Durrant's flax genotrophs, L and S, and flax genotypes, R and M. MDH activity in leaves from just below the inflorescence was higher in the two smaller, sparsely branched plant types, S and M, than in the larger, more branched plant types, L and R. The MDH electrophoretic banding pattern in flax leaf extracts consisted of three major anionic bands, MDH-1, MDH-2, and MDH-3. NoR _m differences were detected between corresponding isozymes of genotypes R and M. For the genotrophs, however, all three bands of S migrated faster than the corresponding bands of L. Codominance was absent in F₁ hybrids; SR _m was dominant for MDH-2 and MDH-3 and LR _m was dominant for MDH-1. The observations suggest that MDHR _m in L and S may be controlled by a modifier locus (or loci). Previous studies indicate that a modifier locus may also control heritable genotrophic differences in peroxidase (PER) and acid phosphates (AP)R _m. The three enzyme systems are compared.The financial assistance of the Natural Sciences and Engineering Research Council of Canada is acknowledged with thanks.     

Alkaloid accumulation in Catharanthus roseus cell suspension cultures fed with stemmadenine

Feeding stemmadenine to Catharanthus roseus cell suspension culture resulted in the accumulation of catharanthine, tabersonine and condylocarpine. Condylocarpine is not an intermediate in the pathway to catharanthine or tabersonine when it is fed to the cultures. The results support the hypothesis that stemmadenine is an intermediate in the pathway to catharanthine and tabersonine.     

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.     

Oil synthesis in vitro in microsomal membranes from developing cotyledons of Linum usitatissimum L.

Microsomal preparations from developing linseed (Linum usitatissimum L.) cotyledons catalyzed i) acyl exchange between acyl-CoA and position 2 of sn-phosphatidylcholine, ii) acylation of sn-glycerol 3-phosphate to yield phosphatidic acid, and iii) the utilisation of phosphatidic acid in the production of diacylglycerol and triacylglycerol. Selectivity studies for C₁₈ acyl species of acyl-CoA indicated a bias for the channelling of oleate to phosphatidylcholine for, presumably, its desaturation, and the utilisation of the polyunsaturated fatty-acid products in the acyl-CoA pool for phosphatidic acid and subsequent triacylglycerol synthesis. The microsomal preparations were capable of returning glycerol backbone with associated acyl components to phosphatidylcholine from diacylglycerol where it may be further enriched with polyunsaturated C₁₈ acids by desaturation. The acyl quality in linolenate-rich oilseeds appears to be under similar control to that found in linoleate-rich species. Present address: To whom the correspondence should be addressed     

Anti-inflammatory oleanane triterpenes from Tripterygium wilfordii cell suspension cultures by fungal elicitation

Summary Treatment of cell suspension cultures of Tripterygium wilfordii with an autoclaved Botrytis sp. homogenate rapidly increased the synthesis of a family of oleanane and friedelane triterpenes, including the antiinflammatory oleanane triterpene 3,22-dihydroxyolean-12-en-29-oic acid. This compound exceeded 30 mg · l^–1 in 13 day elicitations with 12 l bioreactors, in contrast to control levels of less than 5 mg · l^–1. Cell cultures treated with the fungal elicitor provided higher triterpene yields in less time than cultures in a diterpene production medium or whole plants. Elicited production has been developed for commercial application in light of the successful treatment of rheumatoid arthritis with Tripterygium extracts.     

The accumulation and isolation of coniferin from a high-producing cell suspension of Linum flavum L.

Cell suspensions of Linum flavum L. contained large amounts (2 g·l^–1) of the glucoside coniferin which was accumulated endogenously up to 12.4% on a dryweight basis. Callus material contained 5.6%, while in leaves of in-vitro-grown plantlets, the origin of the callus and suspension cultures, no coniferin could be detected. Leaf, callus and suspension material were compared for metabolite accumulation and associated enzyme activities. High coniferin contents corresponded with low 5-methoxypodophyllotoxin levels. A reciprocal relationship between -glucosidase (E.C. 3.2.1.21) activity and coniferin accumulation was found. No relationship between peroxidase (E.C. 1.11.1.7) activity and coniferin accumulation or 5-methoxypodophyllotoxin could be demonstrated. Finally, a rapid and effective isolation procedure for coniferin was developed.Abbreviation HPLC high-performance liquid chromatography This study has been performed within PDI (Plant Disciplines Integrated), a cooperation between the Free University of Amsterdam, TNO-ITC, Zeist and the University of Groningen, The Netherlands. We would like to thank Dr. H.J. Wichers for taking care of mass spectrometric analysis.     

A lipoxygenase-divinyl ether synthase pathway in flax (Linum usitatissimum L.) leaves

Incubation of linoleic acid with an enzyme preparation from leaves of flax (Linum usitatissimum L.) led to the formation of a divinyl ether fatty acid, i.e. (9Z,11E,1'Z)-12-(1'-hexenyloxy)-9,11-dodecadienoic [(omega5Z)-etheroleic] acid, as well as smaller amounts of 13-hydroxy-9(Z),11(E)-octadecadienoic acid. The 13-hydroperoxide of linoleic acid afforded the same set of products, whereas incubations of alpha-linolenic acid and its 13-hydroperoxide afforded the divinyl ether (9Z,11E,1'Z,3'Z)-12-(1',3'-hexadienyloxy)-9,11-dodecadienoic [(omega5Z)-etherolenic] as the main product. Identification of both divinyl ethers was substantiated by their UV, mass-, (1)H NMR and COSY spectral data. In addition to the 13-lipoxygenase and divinyl ether synthase activities demonstrated by these results, flax leaves also contained allene oxide synthase activity as judged by the presence of endogenously formed (15Z)-cis-12-oxo-10,15-phytodienoic acid in all incubations.     

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.     

Methyl jasmonate-induced rosmarinic acid biosynthesis in Lithospermum erythrorhizon cell suspension cultures

Summary A dramatic increase in rosmarinic acid (RA) content in cultured cells of Lithospermum erythrorhizon was observed after their exposure to methyl jasmonate (MJ). Preceding the induced RA accumulation, phenylalanine ammonia-lyase (PAL) and 4-hydroxyphenylpyruvate reductase (HPR) activities increased rapidly and transiently, whereas tyrosine aminotransferase (TAT) activity showed only a slight increase. The elicitation activity of MJ was much higher than that of yeast extract (YE) in terms of the induction of PAL and HPR activities, RA accumulation and incorporation of both ¹⁴C-phenylalanine and ¹⁴C-tyrosine into RA. However, the response of the cultured cells to MJ-treatment was slower than that to YE-treatment.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - LS Linsmaier and Skoog - HPR 4-hydroxyphenylpyruvate reductase - PAL phenylalanine ammonia-lyase - TAT tyrosine aminotransferase - MJ methyl jasmonate - YE yeast extract