Lignin release and photomixotrophism in suspension cultures of Picea abies

The effect of different concentrations of sucrose (0-4%) and of two growth regulators (0–50 μ M 2,4-D and 0–25 μ M kinetin) was tested on growth and chlorophyll content of suspension cultures of Picea abies (L.) Karst. originating from chlorophyllous embryo callus in an elevated CO₂ (2%) atmosphere. A continuous chlorophyllous suspension culture was achieved on a medium containing 2% sucrose and a low level of organic nitrogen (0.25 m M arginine and 0.5 m M glutamine) supplemented with 2,4-D (0.5 μ M ) and kinetin (2.5 μ M ). The same medium with 4% sucrose gave the best growth response, but a negative correlation between chlorophyll level and growth was observed. The chlorophyllous cultures grew slowly in a medium with low (0.5%) sucrose or without any carbohydrate source, suggesting photomixotrophism. A high concentration of kinetin inhibited both growth and chlorophyll synthesis. Release of lignin into the nutrient medium was observed in several experiments, especially in slow-growing cultures supplemented with sucrose. Only a few successive passages of suspensions that produced lignin could be cultured before cell death. The cultures releasing lignin may be unique for studies on synthesis and biodegradation of this very complex compound.     

Use of RAPD patterns for clone verification and in studying provenance relationships in Norway spruce (Picea abies)

 We have used the RAPD technique to analyse samples of Picea abies obtained from an improvement forestry station. Two types of plant material were harvested, the first being clones and the second provenances from various regions. We first checked the clonal identity of elite tree cuttings and clones; some differences in the RAPD patterns resulting from mis-planting or mis-labelling of cuttings were found. We also established a reference library of RAPD fingerprints for 96 clones, which will serve as a reference source in cases of litigation concerning clone identity. The RAPD technique was also used to study the genetic relationship between nine European provenances of Norway spruce. A dendogram was obtained by individual pairwise comparison of 42 RAPD bands, which separated the nine provenances into two major groups, one containing the Nordic provenances (Sweden and Bielorussia) and another the Alpine provenances (France, Austria, Germany and Belgium). The Belgian provenance, which is not indigenous, is most closely related to the German provenance. We conclude that the RAPD technique is a useful tool for forestry stations in managing propagation operations. Received: 15 June 1996 / Accepted: 11 October 1996     

Rhizobial Nod factors stimulate somatic embryo development in Picea abies

 Nod factors are lipochitooligosaccharides (LCOs) secreted by rhizobia. Nod factors trigger the nodulation programme in a compatible host. A bioassay was set up to test how crude (NGR234) and purified (NodS) Nod factors influence cell division and somatic embryogenesis in a conifer, Norway spruce (Picea abies). The Nod factors promoted cell division in the absence of auxin and cytokinin. More detailed studies showed that NodS stimulates development of proembryogenic masses from small cell aggregates and further embryo development. However, stimulation was only observed in low-density cell cultures. Our data suggest that rhizobial Nod factors substitute for conditioning factors in embryogenic cultures of Norway spruce. Received: 20 January 1999 / Revision received: 26 March 1999 / Accepted: 27 April 1999     

The concentrations of Fe, Zn and Co in successive needle age classes of Norway spruce [Picea abies (L.) Karst.]

The concentrations of Fe, Zn and Co were determined in up to five successive needle age classes in 54 individual Norway spruce trees from eight different sites (soil pH 3.1–7.7). Fe concentrations (12–25 μg in needles from the current year) were lower than most published values, due to the removal of surface contamination prior to analysis. Fe showed a significant positive correlation with Al. Successive needle age classes either had constant values or showed an increase for Fe concentrations; individual trees on a given site were rather uniform in their behaviour. Zn concentrations were 19–40 μg/g. On acid sites, they showed a positive correlation with total soil concentrations. The majority of trees showed decreasing Zn concentrations in successive needle age classes, but constant or increasing concentrations were also found; site homogeneity was less than with Fe. Co concentrations differed between trees on a neutral soil (12 ng/g) and on acid soils (41–174 ng/g). They showed a significant positive correlation with Mn needle concentrations. The changes of Co with needle age in most, but not all, trees were similar to those of Zn. The different changes of Fe, Zn and Co with needle age may be due to a different retranslocation. A modest retranslocation of Fe as opposed to a high retranslocation of Zn and Co (in most trees) is consistent with the observed behaviour. Received: 10 May 1999 / Accepted: 8 September 1999     

Development of EST-PCR markers and monitoring their intrapopulational genetic variation in Picea abies (L.) Karst.

Fifteen cDNA sequences are reported for the European coniferous forest tree species Norway spruce [Picea abies (L.) Karst.], including the results of similarity searches in public electronic databases. The sequences were subsequently employed for the design of specific primer pairs and PCR-based amplification of genomic fragments. For seven primer pairs, polymorphic EST-PCR markers were identified among 18 trees. Their mode of inheritance was verified by analysing single-tree offspring and studying segregation among haploid endosperms in comparison to diploid tissue. Codominant inheritance was indicated for six markers, while one marker was apparently dominant. Variation of the six codominant EST markers was tested by genotyping 110 randomly selected trees in a Bavarian Norway spruce population. For comparison, the same trees were genotyped at 18 enzyme coding gene loci. There were 3.33 alleles per locus for EST markers and 3.00 for isoenzyme gene markers. In general, a trend to more even frequency distributions and larger intrapopulational variation, including observed heterozygosities, was indicated more for EST markers than for isoenzyme gene markers. The benefits of these newly developed EST-PCR markers are outlined with respect to population genetics and ecological genetics. Received: 29 April 2000 / Accepted: 25 August 2000     

Nad1 b/c intron polymorphism reveals maternal inheritance of the mitochondrial genome in Picea abies

 Our goal was to determine the mode of inheritance of Picea abies mitochondria. We studied four trees implicated in intraspecific controlled crosses. Based on a PCR-derived technique, one mitochondrial marker, the intron b/c of subunit 1 of NADH dehydrogenase (nad1 b/c), was informative in one of the available crosses. This fragment was sequenced and exhibited several features concordant with the group-II intron, as judged from its sequence evolution and organisation. All of the 96 offspring analysed from the two polymorphic parents present a maternal profile. We conclude that in P. abies the mitochondrial genome is maternally inherited. This result is congruent with the commonly observed pattern of mitochondrial inheritance in the other Picea species studied so far. The existence of mtDNA polymorphic markers that are maternally inherited should now facilitate the study of the population genetic structure of the Norway spruce. Received: 5 December 1998 / Accepted: 28 December 1998     

Image analysis of the stomatal cell walls of Picea abies (L.) Karst. in pure and ozone-enriched air

 The method of image analysis was used to re-investigate previously described structural changes in spruce needles following their exposure to ozone. The changes observed in fumigation experiments were compared with the modifications observed at a site with a similar ozone load. Use was made in each case of needles for which the varying behavioural patterns were known from physiological experiments. In samples with reduced ability to regulate stomatal aperture, the cell walls of the stomatal apparatus showed a reduction in absorptive power at 280 nm. This was interpreted to be a result of delignification of the cell walls in question due to the direct impact of ozone on the surface of the needle. Received: 21 May 1997 / Accepted: 30 June 1997     

Extracellular proteins in embryogenic suspension cultures of Norway spruce (Picea abies)

Embryogenic cell lines of Norway spruce ( Picea abies ) varying in growth habit and morphology were compared as regards profiles of extracellular proteins. Similar proteins were detected in the culture medium by SDS PAGE and in vivo labeling experiments, indicating that the proteins were secreted. Approximately 20 protein bands could be detected in the medium of each cell line. Three of the bands represented glycosylated proteins, as revealed by Concanavalin A staining. Some of the secreted proteins were similar for all tested embryogenic lines of Norway spruce, others were either specific for a group of cell lines or for individual cell lines. A correlation was observed between the morphology of the somatic embryos in a cell line and the presence of secreted proteins. The embryogenic cell lines of Norway spruce can be divided into two main groups. A and B, where A is characterized by somatic embryos with dense embryoheads and B by somatic embryos with loosely aggregated cells in their embryoheads. When proteins secreted from a cell line belonging to group A were added to cell lines belonging to group B, the somatic embryos of the B type developed further and became more similar in morphology to A-type embryos. These observations indicate that cell lines belonging to group A secrete certain proteins to the culture medium that are essential for the development of somatic embryos of Norway spruce.     

Ethylene effects on cambial activity and cell wall formation in hypocotyls of Picea abies seedlings

Ethylene regulation of cell division in the vascular cambium and cell wall formation was studied in hypocotyls of Norway spruce ( Picea abies [L.] Karst.) seedlings. Cuttings from 6-week-old seedlings were placed in water culture to which compounds affecting the synthesis and action of ethylene were added. After a 3-week treatment period, growth, ethylene production, morphology and cell wall composition of the hypocotyls were determined. Addition of high concentrations of the potent ethylene releasing agent 2-chloroethylphosphonic acid (ethrel), which increased ethylene emission by more than twice compared to control plants, inhibited the expansion of xylem cells while stimulating the incorporation of cell wall material, especially cellulose. Addition of small amounts of ethrel, which slightly stimulated ethylene emission, led to increases in the size of xylem cells, the amount of phloem tissue and the number of intercellular spaces in the cortex, and thus to increased hypocotyl diameter. However, no significant change in cell wall composition was detected. When ethylene production was decreased by adding Co²⁺ to the nutrient solution, differentiation of new xylem was disturbed, but the rate of cell division was not affected. Although the incorporation of cell wall material was inhibited, the proportions of lignin and cellulose in the wall appeared to remain unchanged. Silver ions stimulated the expansion of both xylem and cortex cells, but had no significant effect on cell wall formation. We conclude that ethylene has a role in regulating the incorporation of wall carbohydrates.     

Characterization of wall-bound invertase isoforms of Picea abies cells and regulation by ectomycorrhizal fungi

In culture, the ectomycorrhiza-forming fungi Amanita muscaria (Pers. ex Fries) Hock. and Hebeloma crustuliniforme (Bull. ex Fries) Quel. only grow on media with glucose or fructose but not with sucrose as sole carbohydrate source. This is due to their lack of wall-bound invertase activity. Therefore, utilization of sucrose by the fungi within a mycorrhizal association is believed to depend on the wall-bound invertase activity of the host. This enzyme activity was studied in the apoplast of suspension cultured cells of Picea abies (L.) Karst. An ionically and a tightly wall-bound isoform of acid invertase were found that function as β-d -fructofuranoside-fructohydrolases (EC 3.2.1.26). The ionically bound enzyme could be easily released from walls of intact cells with buffer of high ionic strength. In its native form, the ionically bound invertase isoform is a monomeric protein with a molecular mass of 61 kDa, as determined by gel filtration and SDS-PAGE. Glycoprotein nature of the enzyme was demonstrated with antibodies directed against the digoxigenin-labeled protein. The K_m values of both enzymes for sucrose, their natural substrate, are relatively high (ionically bound invertase K_m= 16 mM, tightly bound invertase K_m= 8.6 mM). Activity of both wall-bound invertase isoforms strongly depends on the apoplastic pH. They have a narrow pH-optimum and exhibit highest activity at pH 4.5. with elevated activity between pH 4.5 and 6.0. Furthermore, fructose acts as competitive inhibitor of both isoforms, whereas glucose is not inhibitory. Unloading of sucrose from host cells to the apoplastic interface of the Hartig net in ectomycorrhizae appears to depend on the rate of hydrolysis by the wall-bound invertase of the host. Since the activity of the plant invertase depends on the actual pH value and the fructose concentration in the mycorrhizal interface, we suggest that the fungus can actively influence the activity of the plant invertase by acidification of the cell wall and by fructose uptake. Thus, the fungus itself can regulate its own supply of glucose and fructose.     

Movement and possible metabolism of ethylene in dormant Picea abies

Ethylene or ethane was added to and sampled from the base, middle and top of the stem of intact Picea abies (L.) Karst. using microdialysis probes. The endogenous concentrations of ethylene and ethane in all positions were below 5 × 10^–9 mol l^–1 throughout the experimental period. Applied ethylene or ethane was always detected in probes above the probe used for the application, never below. If increased ethylene or ethane concentrations were detected, the ethylene concentrations initially increased but thereafter decreased to the basal level, whereas levels of ethane steadily increased. It is proposed that the maintainance of low ethylene levels is important in the regulation of wood formation.     

The cinnamyl alcohol dehydrogenase gene structure in Picea abies (L.) Karst.: genomic sequences, Southern hybridization, genetic analysis and phylogenetic relationships

 Based on PCR technologies, we have isolated three genomic cinnamyl alcohol dehydrogenase (CAD) clones from Norway spruce, Picea abies (L.) Karst., revealing about 99% identity within their protein coding regions. All clones contain five introns with an identity of 97–100% for intervening sequences II, III and IV, whereas intron V sequences revealed only 87–89% identity. Intron I sequences share an identity of 85–98% among all three clones. Intron IV is only present in Norway spruce and not found in published genomic CAD sequences of angiosperms. Tandem repeats between 24 and 49 bp were discovered within intervening sequences I and V. Southern hybridization of seedling DNA and PCR-based intron analyses using diploid leaf buds and haploid megagametophytes indicate the existence of a small CAD gene family within the spruce genome, consisting of at least two loci. Evolutionary analyses of CAD encoding sequences using distance matrix- and parsimony-based methods revealed that CADs from angiosperms form a clade distinct from those of gymnosperms. Confirmed by maximal bootstrap values of 100%, a gene duplication gave rise to two different groups of angiospermous CADs and this duplication may have occurred in an early stage of angiosperm radiation, certainly before the separation of the Dilleniidae and Rosidae lineages. Phylogenetic investigations suggest angiosperm CAD II sequences to have evolved more rapidly than angiosperm CAD I genes. On the other hand, CAD gene evolution appears to be significantly slower in conifers than in angiosperms. Received: 27 February 1998 / Accepted: 22 April 1998     

Metabolism of tritiated gibberellin A1 in seedlings of Norway spruce, Picea abies

Five-week-old seedlings of Norway spruce, Picea abies (L.) Karst., metabolized 1,2-/³H/-gibberellin A₁ into a single major compound chromatographically similar to gibberellin A₈. The conversion rate exceeded 10% within the 24-h incubation period.     

Biological activity, identification and quantification of gibberellins in seedlings of Norway spruce (Picea abies) grown under different photoperiods

Short photoperiod induces growth cessation in seedlings of Norway spruce ( Picea abies (L.] Karst.). Application of different gibberellins (GAS) to seedlings growing under a short photoperiod show that GA₉ and GA₂₀ can not induce growth. In contrast application of GA, and GA₄ induced shoot elongation. The results indicate that 3β-hydroxylation of GA₉ to GA₄ and of GA₂₀ to GA₁ is under photoperiodic control. To confirm that conclusion, both qualitative and quantitative analyses of endogenous GAs were performed. GA₁, GA₃, GA₄, GA₇, GA₉, GA₁₂, GA₁₅, GA₁₅, GA₂₀, GA₂₉, GA₃₄ and GA₅₁ were identified by combined gas chromatography-mass spectrometry in shoots of Norway spruce seedlings. The effect of photoperiod on GA levels was determined by using deuterated and ¹⁴C-labelled GAs as intermal standards. In short days, the amounts of GA₉, GA₄ and GA₁ are less than in plants grown in continuous light. There is no significant difference in the amounts of GA₃, GA₁₂, and GA₂₀ between the different photoperiods. The lack of accumulation of GA₉ and GA₂₀ under short days is discussed.     

In vitro phyllomorphic regeneration of shoot buds and shoots in Picea abies

Needles (10–15 mm) of frost-hardened 20–22-week-old (physiological age equivalent to 1 year) plants of Picea abies L. excised just after flushing, were induced to form adventitious shoot buds and shoots on media supplemented with BAP (6-benzylaminopurine) and NAA (1-napthaleneacetic acid). The addition of nanomolar concentrations (0.5–50) of NAA combined with 1–10 μM BAP considerably stimulated formation of pseudobulbils on the basal to mid-part of the needle axis, as well as their subsequent development into shoot buds and shoots. On a medium containing 10 μM BAP, pseudobulbils that formed at the needle base did not develop further, but became necrotic and died with the omission of NAA. With 5 μM BAP + 50 nM NAA the initial phase of development was slow, but later showed good response and up to 22% of the needles produced shoot buds. Two to three shoots per needle could be excised and subcultured individually onto fresh media. It is concluded that the level of endogenous auxin decreases progressively from the needle's base to its tip, so that that concentration of exogenous auxin (50 nM NAA) which promotes pseudobulbil and shoot-bud formation part-way along the needle axis, simultaneously inhibits their induction at the needle base.     

Interaction between atmospheric and pedospheric nitrogen nutrition in spruce (Picea abies L. Karst) seedlings

The effect of NO₂ fumigation on root N uptake and metabolism was investigated in 3-month-old spruce (Picea abics L. Karst) seedlings. In a first experiment, the contribution of NO₂ to the plant N budget was measured during a 48 h fumigation with 100mm³m^?3 NO₂. Plants were pre-treated with various nutrient solutions containing NO₂ and NH₄⁺, NO₃^? only or no nitrogen source for 1 week prior to the beginning of fumigation. Absence of NH₄⁺ in the solution for 6d led to an increased capacity for NO₃^? uptake, whereas the absence of both ions caused a decrease in the plant N concentration, with no change in NO₃^? uptake. In fumigated plants, NO₂ uptake accounted for 20–40% of NO₃^? uptake. Root NO₃^? uptake in plants supplied with NH₄⁺plus NO₃^? solutions was decreased by NO₂ fumigation, whereas it was not significantly altered in the other treatments. In a second experiment, spruce seedlings were grown on a solution containing both NO₂ and NH₄⁺ and were fumigated or not with 100mm³m^?3 NO₂ for 7 weeks. Fumigated plants accumulated less dry matter, especially in the roots. Fluxes of the two N species were estimated from their accumulations in shoots and roots, xylem exudate analysis and ¹⁵N labelling. Root NH₄⁺ uptake was approximately three times higher than NO₃^? uptake. Nitrogen dioxide uptake represented 10–15% of the total N budget of the plants. In control plants, N assimilation occurred mainly in the roots and organic nitrogen was the main form of N transported to the shoot. Phloem transport of organic nitrogen accounted for 17% of its xylem transport. In fumigated plants, neither NO₃^? nor NH₄⁺ accumulated in the shoot, showing that all the absorbed NO₂ was assimilated. Root NO₃^? reduction was reduced whereas organic nitrogen transport in the phloem increased by a factor of 3 in NO₂-fimugated as compared with control plants. The significance of the results for the regulation of whole-plant N utilization is discussed.     

Ethylene effects on peroxidases and cell growth patterns in Picea abies hypocotyl cuttings

The effect of 2-chloroethylphosphonic acid (ethrel) on cell growth patterns and per-oxidase activity (EC 1.11.1.7) and location in young Norway spruce cuttings ( Picea abies [L.] Karst.) was investigated. The peroxidase activity in a fraction containing soluble and membrane bound enzymes show a diurnal variation, with decreased activity during the light period and a corresponding increase during the following dark period. The decrease during the day could to some extent be counteracted by treatment with ethrel. It appears that ethrel affects only peroxidases in the isolated membrane fraction, since peroxidases bound to the cell wall were not affected by ethrel. In vitro experiments indicated that the hydrophobicity of soluble peroxidases was increased by treatment with ethylene. Cytochemical localization of peroxidase activity in differentiating tracheids revealed a clear ethrel-induced increase in the tonoplast. It appears that ethylene affects soluble peroxidases in vivo in such a way that they are directed to a more hydrophobic environment, like the tonoplast. Treatment with ethrel also changed the appearance of the rough endoplasmic reticulum (ER) and Golgi apparatus. Dilated ER cisternae were observed on electron micrographs, as a result of treatment with ethrel. The number of vesicles produced by the Golgi apparatus and also the amount of vesicles fusing with the plasma membrane in secondary-wall-forming tracheids increased considerably. The results clearly indicate that the stimulatory effect of ethylene in spruce seedlings on lignification and cell wall formation, is due to a general stimulation on both synthesis, transport and secretion of cell wall material and not on a stimulation of peroxidase activity as reported for other species.     

Bacterial endophytes from seeds of Norway spruce (Picea abies L. Karst)

Endophytic bacteria from wooden plants and especially seed-associated endophytes are not well studied. Fresh seeds collected from four Norway spruce trees (Picea abies) from different locations in the Slovene subalpine region were surface-sterilised and dissected into a seed coat, embryo and endosperm. The presence of endophytes was detected by culturing methods and by direct amplification of the eubacterial 16S rDNA gene. Both approaches identified bacteria from genera Pseudomonas and Rahnella in the Norway spruce seeds. Both are known plant-associated bacteria with growth-promoting properties and biological control potential. We suggest that plant seeds could serve as a vector for transmission of beneficial bacteria.     

ATP-induced sucrose efflux from red-beet tonoplast vesicles

 Sucrose efflux from the vacuole of mobilizing red-beet (Beta vulgaris L.) hypocotyl cells was investigated using purified tonoplast vesicles. Tonoplast vesicle purity was assured by the immunoreactivity to antibodies raised against the vacuolar ATPase and by the strong inhibition exhibited by the H⁺-ATPase to bafilomycin-A and NO₃ ⁻. Inhibition of the H⁺-ATPase by vanadate and azide was negligible. Sucrose was loaded into tonoplast vesicles by using the pH-jump method of energization. Addition of ATP to sucrose-loaded vesicles in the presence of bafilomycin-A resulted in efflux of a significant amount of sucrose. During ATP-induced sucrose efflux, bafilomycin-insensitive ATPase activity increased significantly with no increase in H⁺-translocating activity. The additional bafilomycin-A insensitive ATPase activity observed in sucrose-loaded vesicles was completely inhibited by vanadate as was the efflux of sucrose. Similar to vanadate, thapsigargin was also inhibitory to sucrose efflux and to the bafilomycin-A insensitive ATPase activity. The data indicate that vacuolar sucrose can be actively mobilized by a specific ATP-dependent efflux mechanism. Received: 12 October 1999 / Accepted: 18 November 1999