In Vivo and in Vitro Evidence for Biochemical Coupling of Reactions Catalyzed by Lysophosphatidylcholine Acyltransferase and Diacylglycerol Acyltransferase

Seed oils of flax (Linum usitatissimum L.) and many other plant species contain substantial amounts of polyunsaturated fatty acids (PUFAs). Phosphatidylcholine (PC) is the major site for PUFA synthesis. The exact mechanisms of how these PUFAs are channeled from PC into triacylglycerol (TAG) needs to be further explored. By using in vivo and in vitro approaches, we demonstrated that the PC deacylation reaction catalyzed by the reverse action of acyl-CoA:lysophosphatidylcholine acyltransferase (LPCAT) can transfer PUFAs on PC directly into the acyl-CoA pool, making these PUFAs available for the diacylglycerol acyltransferase (DGAT)-catalyzed reaction for TAG production. Two types of yeast mutants were generated for in vivo and in vitro experiments, respectively. Both mutants provide a null background with no endogenous TAG forming capacity and an extremely low LPCAT activity. In vivo experiments showed that co-expressing flax DGAT1-1 and LPCAT1 in the yeast quintuple mutant significantly increased 18-carbon PUFAs in TAG with a concomitant decrease of 18-carbon PUFAs in phospholipid. We further showed that after incubation of sn-2-[¹⁴C]acyl-PC, formation of [¹⁴C]TAG was only possible with yeast microsomes containing both LPCAT1 and DGAT1-1. Moreover, the specific activity of overall LPCAT1 and DGAT1-1 coupling process exhibited a preference for transferring ¹⁴C-labeled linoleoyl or linolenoyl than oleoyl moieties from the sn-2 position of PC to TAG. Together, our data support the hypothesis of biochemical coupling of the LPCAT1-catalyzed reverse reaction with the DGAT1-1-catalyzed reaction for incorporating PUFAs into TAG. This process represents a potential route for enriching TAG in PUFA content during seed development in flax.     

An untargeted liquid chromatography–mass spectrometry‐based workflow for the structural characterization of plant polyesters

Cell wall localized heterogeneous polyesters are widespread in land plants. The composition of these polyesters, such as cutin, suberin, or more plant‐specific forms such as the flax seed coat lignan macromolecule, can be determined after total hydrolysis of the ester linkages. The main bottleneck in the structural characterization of these macromolecules, however, resides in the determination of the higher order monomer sequences. Partial hydrolysates of the polyesters release a complex mixture of fragments of different lengths, each present in low abundance and therefore are challenging to structurally characterize. Here, a method is presented by which liquid chromatography–mass spectrometry (LC‐MS) profiles of such partial hydrolysates are searched for pairs of related fragments. LC‐MS peaks that show a mass difference corresponding to the addition of one or more macromolecule monomers were connected in a network. Starting from the lowest molecular weight peaks in the network, the annotation of the connections as the addition of one or more polyester monomers allows the prediction of consecutive and increasingly complex adjacent peaks. Multi‐stage MS (MSn) experiments further helped to reject, corroborate, and sometimes refine the structures predicted by the network. As a proof of concept, this procedure was applied to partial hydrolysates of the flax seed coat lignan macromolecule, and allowed to characterize 120 distinct oligo‐esters, consisting of up to six monomers, and containing monomers and linkages for which incorporation in the lignan macromolecule had not been described before. These results showed the capacity of the approach to advance the structural elucidation of complex plant polyesters.     

‘范妮’亚麻高频率体细胞胚胎发生及组织学观察

以亚麻品种‘范妮’的子叶和下胚轴为外植体,分别接种在不同组合的植物生长调节剂的MB(MS培养基的无机盐加B5培养基的维生素)培养基上培养,结果所有使用的培养基都能诱导出愈伤组织,但只有在附加了0.5~4.0 mg.L-12,4-D培养基上才能诱导出胚性愈伤组织。在胚性愈伤组织和体胚诱导中,源于下胚轴的材料均比源于子叶的材料所需的2,4-D浓度低。在培养基中,单独使用2,4-D诱导体胚的效果明显好于使用其它植物生长调节剂的组合,源于子叶和下胚轴的体胚诱导数分别达到了224个/g和265个/g。组织学观察发现有大量正常的球形胚、心形胚、鱼雷胚和少量子叶胚,还有少量畸形胚。本文为‘范妮’与其它麻类的体细胞杂交育种、‘范妮’变异无性系的筛选及相关的植物基因工程和细胞工程研究奠定了一定的工作基础。     

Development of Linum flavum hairy root cultures for production of coniferin

Linum flavum hairy roots were initiated from leaf discs using Agrobacterium rhizogenes strains LBA9402 and TR105 though two other strains, 15834 and A4, were relatively ineffective for induction. Significant variation in coniferin accumulation was observed between hairy root lines originating from different L. flavum seedlings and/or A. rhizogenes strains. Coniferin reached 58 mg g^–1 dry wt by culturing the roots in Linsmaier and Skoog (LS) medium with 2,4-dichlorophenoxyacetic acid and naphthaleneacetic acid as growth regulators.     

Flax anther culture: effect of genotype,cold treatment and media

We report on screening of wide range of flax cultivars for androgenic response and on testing of induction conditions for flax (Linum usitatissimum L.) anther culture and plant regeneration. Anthers were cultured on four different media: Mo, N6, MS and N&N supplemented with various combinations of growth regulators. The induction of callus formation from cultured anthers was the highest on N6 (with cultivar PR FGL 77 – 12 %) and N&N media (with cultivar Carolin – 2.8 %), preferentially after cold pretreatment (7days at 8 °C). Shoots were formed on calli derived from the microspores inside the cultured anthers on media N&N and N6 supplemented with 1mgl^–1 zeatin or 1mgl^–1BAP + 1mgl^–1NAA, respectively and elongated on MS medium supplemented with 2mgl^–1 zeatin. The highest number of shoots (120) was observed with cultivar Red Wing. Shoots were rooted on MS medium supplemented with 2mgl^–1IAA. Our experiments resulted in total in 62 % anther response and 155 plants regenerated and transferred into soil.     

Mechanisms and control of rapid genomic changes in flax

BACKGROUND AND AIMS: The nuclear DNA of certain varieties of flax (Linum usitatissimum) can vary within a single generation when the plants are grown under specific environmental conditions. This review details the genomic variations that have been identified and associated with this environmental response. CONCLUSIONS: The variation occurs across the whole spectrum of sequence repetition and has been shown to occur in the highly repeated, middle repetitive and low copy number sequences. Although the variation has been shown to be spread throughout the genome it does not occur at random, as similar molecular events have been shown to occur repeatedly. The changes in two labile regions in the nucleus, the ribosomal RNA genes and a site-specific insertion event, have been shown to occur within the period of vegetative growth and over a relatively short period of that growth. The gradual change in total nuclear DNA that has been described would then need to have arisen through an accumulation of changes occurring over the whole, or most of the, period of growth prior to flowering. The polymorphisms that result from these rapidly occurring genomic events have also been observed in many other flax and linseed varieties as well as in the wild progenitors of flax.     

Enhancement of lignan biosynthesis in suspension cultures of <Emphasis Type="Italic">Linum nodiflorum</Emphasis> by coronalon,indanoyl-isoleucine and methyl jasmonate

The effect of the two synthetic elicitors coronalon and indanoyl-isoleucine and of methyl jasmonate (MeJA) on the accumulation and biosynthesis of lignans by cell suspension cultures of Linum nodiflorum (Linaceae) was investigated. The production of 6-methoxypodophyllotoxin (MPTOX) could be increased more than tenfold, the maximal content reaching up to over 2.5% of the cell dry weight. The highest yield was achieved by administering 50 μM of the synthetic elicitors on the fourth day and extracting the products on the tenth day of the culture period. An additional lignan accumulated in elicitor-treated cultures. Its structure was elucidated by extensive 1D and 2D NMR measurements, revealing its identity as 5′-demethoxy-MPTOX (5′-dMPTOX). Its average content amounted up to over 5% of the cell dry weight. Growth was only slightly affected by the addition of the elicitors. Methyl jasmonate exerted a moderate stimulating effect on the L. nodiflorum cells with MPTOX and 5′-dMPTOX contents going up to 1.4 and 2.1% of the cell dry weight, respectively. The activities of deoxypodophyllotoxin 6-hydroxylase and β-peltatin 6-O-methyltransferase, two enzymes involved in MPTOX biosynthesis, were increased up to 21.9-fold and 14.6-fold, respectively, in the treated cultures.     

Identification and partial characterization of proteins and proteoglycans encrusting the secondary cell walls of flax fibres

 Four proteins were isolated from depectinised elementary fibres of flax (Linum usitatissimum L.), using either alkali or cellulase digestion treatments. All the four proteins were characterized by a deficiency or low contents of hydroxyproline and by high levels of glutamic acid/glutamine and/or aspartic acid/asparagine. The two proteoglycans solubilized with cellulase strongly reacted with β-glucosyl Yariv reagent but not with α-glucosyl Yariv reagent and contained appreciable amounts of alanine, glycine, serine and threonine, suggesting a relationship with cell wall hydroxyproline-deficient arabinogalactan-proteins. The two alkali-extracted proteins did not show any reaction with β-glucosyl Yariv dye. Due to the harsh treatment, they might only partially represent the original proteins. Due to its high level of glycine (41%), one of these proteins might be classified as a glycine-rich protein. The latter polypeptide, of low molecular molar mass, contained 14.6% leucine and might consist of a domain related to leucine-rich proteins. The data show that these proteins and arabinogalactan-protein-like proteoglycans were strongly associated with the secondary walls of flax fibres. Their presence in small amounts (0.1–0.4%), raises the problem of their putative structural role. Received: 22 October 1999 / Accepted: 17 January 2000     

Variation and evolution inLinum tenuifolium (Linaceae)

The phenetic variation inL. tenuifolium s.l. was assessed using multivariate analyses of 27 characters found to be variable within the species. These results are discussed and interpreted in the context of the reproductive biology, chromosome number and ecological responses of the taxa. Evidence suggests that both the self-compatible and tetraploid races were derived independently from a self-incompatible, diploid type. Although no taxonomic scheme is compatible with all of the evidence presented, the study supports a recent decision to recognize four taxa at subspecific level.     

亚麻下胚轴转化系统的建立

亚麻是重要的纤维和油料作物,建立简易有效的转化系统,利用农杆菌介导的方法引入有用的外源基因具有十分重要意义。已知不同亚麻品种被根癌农杆菌(Agrobacterium tumefaciens)和发根农杆菌(Agrobacterium rhizogenes)感染的能力差别很大(Zhan XC等 1989)。植物的基因型和农