Liisa Kaarina Simola

Expression profiling of the lignin biosynthetic pathway in Norway spruce using EST sequencing and real-time RT-PCR.

Lignin biosynthesis is a major carbon sink in gymnosperms and woody angiosperms. Many of the enzymes involved are encoded for by several genes, some of which are also related to the biosynthesis of other phenylpropanoids. In this study, we aimed at the identification of those gene family members that are responsible for developmental lignification in Norway spruce (Picea abies (L.) Karst.). Gene expression across the whole lignin biosynthetic pathway was profiled using EST sequencing and quantitative real-time RT-PCR. Stress-induced lignification during bending stress and Heterobasidion annosum infection was also studied. Altogether 7,189 ESTs were sequenced from a lignin forming tissue culture and developing xylem of spruce, and clustered into 3,831 unigenes. Several paralogous genes were found for both monolignol biosynthetic and polymerisation-related enzymes. Real-time RT-PCR results highlighted the set of monolignol biosynthetic genes that are likely to be responsible for developmental lignification in Norway spruce. Potential genes for monolignol polymerisation were also identified. In compression wood, mostly the same monolignol biosynthetic gene set was expressed, but peroxidase expression differed from the vertically grown control. Pathogen infection in phloem resulted in a general up-regulation of the monolignol biosynthetic pathway, and in an induction of a few new gene family members. Based on the up-regulation under both pathogen attack and in compression wood, PaPAL2, PaPX2 and PaPX3 appeared to have a general stress-induced function.

Changes in Polyamine Metabolism during Somatic Embryogenesis in Picea abies

Summary The concentrations of free, conjugated and bound polyamines (putrescine and spermidine) and the activity of a key enzyme, [S-adenosylmethionine decarboxylase (SAMdc)], which participates in the biosynthesis of spermidine, were studied in embryogenic and non-embryogenic callus cultures of Picea abies (L.) Karst. When the calli were transferred on ABA (10 gM) containing maturation media, where proembryos in embryogenic callus develop to mature embryos, both tissues contained high levels of free and conjugated putrescine. After 5 days the putrescine levels decreased in both tissues and did not rise thereafter. The total spermidine level (free, conjugated and bound) was nearly four-fold higher in the embryogenic callus in comparison to the non-embryogenic tissue during the development of globular embryos in the embryogenic callus. In our cultures a positive correlation was found between spermidine concentration and SAMdc-activity. If methylglyoxal bis(guanylhydrazone) (0.1 mM), an inhibitor of SAMdc, was added to the nutrient medium of embryogenic cultures, the levels of total spermidine decreased to nearly half, although further development of somatic embryos was not inhibited.

Characterization of basic p-coumaryl and coniferyl alcohol oxidizing peroxidases from a lignin-forming Picea abies suspension culture

A Norway spruce (Picea abies) tissue culture line that produces extracellular lignin into the culture medium has been used as a model system to study the enzymes involved in lignin polymerization. We report here the purification of two highly basic culture medium peroxidases, PAPX4 and PAPX5, and isolation of the corresponding cDNAs. Both isoforms had high affinity to monolignols with apparent Km values in µM range. PAPX4 favoured coniferyl alcohol with a six-fold higher catalytic efficiency (Vmax/Km) and PAPX5 p-coumaryl alcohol with a two-fold higher catalytic efficiency as compared to the other monolignol. Thus coniferyl and p-coumaryl alcohol could be preferentially oxidized by different peroxidase isoforms in this suspension culture, which may reflect a control mechanism for the incorporation of different monolignols into the cell wall. Dehydrogenation polymers produced by the isoforms were structurally similar. All differed from the released suspension culture lignin and milled wood lignin, in accordance with previous observations on the major effects that e.g. cell wall context, rate of monolignol feeding and other proteins have on polymerisation. Amino acid residues shown to be involved in monolignol binding in the lignification-related Arabidopsis ATPA2 peroxidase were nearly identical in PAPX4 and PAPX5. This similarity extended to other peroxidases involved in lignification, suggesting that a preferential structural organization of the substrate access channel for monolignol oxidation might exist in both angiosperms and gymnosperms.

Lignins released from Picea abies suspension cultures : true native spruce lignins ?

Abstract Suspension-cultured cells of Picea abies release lignin in the nutrient medium. Chemical analyses, 1 H and 13 C NMR spectroscopy of this material show that they represent carbohydrate-free guaiacyl type lignins with a high proportion of cinnamyl alcohol side chains a somewhat lower methoxyl content than milled wood lignin from spruce. The molecular weight distribution determined by HPSEC gave a weight average M r of 14 200 and a number average weight close to 4000. These are the first conifer lignin samples to be characterized without chemical or mechanical degradation.

Labelling of a lignin from suspension cultures of Picea abies

Abstract The lignin released by suspension cultures of Picea abies into the culture medium was labelled with 13 C by adding uniformly-labelled glucose to the culture. The successful labelling (the lignin contained ca 20% 13 C) greatly facilitates the acquisition of detailed and reliable structural information using NMR and makes it possible to carry out three dimensional HMQC-HOHAHA experiments. These spectral data were compared with similar data from lignin that had been isolated from cell walls of intact pine ( Pinus sylvestris ) and from a polymer that had been prepared in vitro by oxidation of labelled coniferyl alcohol. Differences in chemical structure have been found that are of importance for the elucidation of the chemical processes involved in the deposition of lignin in the walls of lignifying tissues.

Changes in the Subcellular Organization of Endosperm and Radicle Cells of Picea abies during Germination

Summary Protein bodies and spherosomes are the main storage structures seen in the cells of dry seeds of Picea abies (L.) Karsten . Small initials of plastids or mitochondria are found in the cytoplasm, but no amyloplasts, dictyosomes or ER. The protein bodies vary greatly in electron-density, especially in the endosperm. They contain some carbohydrate, obviously glycoprotein, and may have a crystalloid. The imbibed protein bodies of endosperm cells usually have one large globoid cavity. Proteolysis is more rapid than lipolysis, but both these processes and the development of new cell organelles are more rapid in the radicle than in the endosperm. At a late stage of germination mitochondria, microbodies (glyoxysomes), ER and proplastids develop in the endosperm cells before these degenerate. Microbodies are very rare in the embryo. In its lipid metabolism, the embryo seems to differ from the endosperm.

The Effect of Several Amino Acids and some Inorganic Nitrogen Sources on the Growth of Drosera rotundifolia in Long- and Short-Day Conditions

Summary The nitrogen metabolism of a carnivorous plant, Drosera rotundifolia L., has been studied in long-day (18 h light) and short-day (12 h light) conditions in aseptic culture. Growth was clearly weaker in short-day conditions but the responses to the nitrogen sources were about the same in the two daylengths. Organic nitrogen was not necessary for flowering in long-day lengths and none of the amino acids induced flowering in short-day lengths. NH 4 NO 3 (1.25 raM) was utilized more effectively than NH 4 Cl (2.5 mM). D. rotundifolia is not more tolerant towards amino acids than other vascular plants. Only arginine was used nearly as effectively as 4 NO 3 (2.5 mM). Aspartic and glutamic acids gave only weak growth. Most of the amino acids tested were not used when given as the only nitrogen source (proline, hydroxyproline, valine, methionine, leucine, isoleucine, lysine and alanine). A low concentration (0.1 mM) of hydroxyproline inhibited the utilization of NH 4 + ions but interfered less with the utilization of NO 3 - ions and very little with that of arginine.

The effect of some non-protein amino acids on pollen germination and pollen-tube growth in five species of the Vicieae.

SummaryThe effects of canavanine, α,γ-diaminobutyric acid, homoarginine and lathyrine on the germination of pollen and on in-vitro growth of pollen tubes were studied in the following species: Lathyrus niger, L. silvestris, Vicia unijuga, Pisum sativum and Cicer arietinum.The effects of these non-protein amino acids depended on their quantity and on the plant species. Every amino acid had a promoting effect on germination and growth at some concentration in some species. Inhibition or promotion of pollen germination and pollen-tube growth were usually parallel. The stronger influence of some amino acid on growth than on germination may be due to slow penetration of the acid into the cell.Homoarginine and lathyrine had a promoting effect at all concentrations in L. niger, a species in which these amino acids occur naturally. In most other species they had, if anything, a very slight inhibitory effect, α,γ-Diaminobutyric acid and canavanine had the strongest inhibitory effects on the species studied. It seems possible that these amino acids are antimetabolites of common amino acids.It is obvious that non-protein amino acids can form effective hybridization barries, although the conditions in nature are more complex than in vitro. The ability to synthesize a new amino acid may therefore be of evolutionary significance in the isolation of new species and genera.

Changes in the activity of several enzymes during root differentiation in cultured cells of Atropa belladonna

Summary Roots were effectively formed by root callus of Atropa belladonna in suspension culture in media containing i. α-naphthoxyacetic acid (NOA 4 ppm) and α-naphthaleneacetic acid (NAA 0.5 ppm) ii. NAA (0.5 ppm). Several enzyme activities estimated separately from roots, from clumps with the roots detached, and from undifferentiated clumps were compared with the activity in an undifferentiated suspension (NAA 2 ppm) of the same age. Corresponding differences in levels of enzyme activity were found in different parts of the cultures irrespective of the type of hormonal induction. For example, roots had higher phosphatase, perioxidase and ribonuclease activities than clumps but glutamate: oxaloacetate transaminase and alanine aminopeptidase activities were higher in clumps with roots than in other parts.

Feeding experiments using suspension cultures of Atropa belladonna; limiting steps in the biosynthesis of tropane alkaloids.

Abstract Relatively low levels of tropane alkaloid precursors (tropic acid, tropinone and tropanol) and alkaloids (hyoscyamine and scopolamine) were fed to suspension cultures of Atropa belladonna with repressed alkaloid synthesis to study the transport of these compounds into the cells, to detect possible limiting steps in the biosynthetic pathway and to find ways of stimulating alkaloid production. At the end of the experiments, the cell levels of tropic acid and tropinone had reached the initial nutrient medium concentrations, while cell levels of tropanol, hyoscyamine and scopolamine were high, suggesting an active uptake mechanism. Increased intracellular precursor levels did not forward the biosynthesis of the tropane alkaloids.