Compositional analysis of essential oil and solvent extracts of Norway spruce sprouts by ultrahigh-resolution mass spectrometry.

INTRODUCTION: Coniferous trees, especially their needles and bark, are a rich source of bioactive compounds. The developing needles of Norway spruce (Picea abies), also known as spruce sprouts, are enriched with vitamin C and other antioxidants, and thus they are used as a dietary supplement and have been traditionally used to treat various inflammatory disorders such as rheumatism and gout. Their chemical composition is only limitedly known, however. OBJECTIVES: The main objective of this work was to have a deeper understanding on the chemical composition of spruce sprouts to assess their full potential in different pharmaceutical, nutraceutical, or technochemical applications. MATERIALS AND METHODS: Ultrahigh-resolution Fourier-transform ion cyclotron (FT-ICR) mass spectrometry, coupled to direct-infusion electrospray ionisation (ESI) or atmospheric pressure photoionisation (APPI) techniques, was used for in-depth compositional analysis of solvent extracts and essential oil of spruce sprouts. RESULTS: A combined use of ESI and APPI techniques offered a great complementary insight into the rich chemistry of different spruce sprout extracts, allowing detection of thousands of chemical constituents with over 200 secondary metabolites tentatively identified. These compounds belonged to different classes such as organic acids, terpenes, flavonoids, stilbenes, sterols, and nitrogen alkaloids. CONCLUSION: Spruce sprouts have a complex metabolite profile that differs considerably from that of the old, developed needles.

Fate of Antioxidative Compounds within Bark during Storage: A Case of Norway Spruce Logs.

Softwood bark is an important by-product of forest industry. Currently, bark is under-utilized and mainly directed for energy production, although it can be extracted with hot water to obtain compounds for value-added use. In Norway spruce (Picea abies [L.] Karst.) bark, condensed tannins and stilbene glycosides are among the compounds that comprise majority of the antioxidative extractives. For developing feasible production chain for softwood bark extractives, knowledge on raw material quality is critical. This study examined the fate of spruce bark tannins and stilbenes during storage treatment with two seasonal replications (i.e., during winter and summer). In the experiment, mature logs were harvested and stored outside. During six-month-storage periods, samples were periodically collected for chemical analysis from both inner and outer bark layers. Additionally, bark extractives were analyzed for antioxidative activities by FRAP, ORAC, and H2O2 scavenging assays. According to the results, stilbenes rapidly degraded during storage, whereas tannins were more stable: only 5-7% of the original stilbene amount and ca. 30-50% of the original amount of condensed tannins were found after 24-week-storage. Summer conditions led to the faster modification of bark chemistry than winter conditions. Changes in antioxidative activity were less pronounced than those of analyzed chemical compounds, indicating that the derivatives of the compounds contribute to the antioxidative activity. The results of the assays showed that, on average, ca. 27% of the original antioxidative capacity remained 24 weeks after the onset of the storage treatment, while a large variation (2-95% of the original capacity remaining) was found between assays, seasons, and bark layers. Inner bark preserved its activities longer than outer bark, and intact bark attached to timber is expected to maintain its activities longer than a debarked one. Thus, to ensure prolonged quality, no debarking before storage is suggested: outer bark protects the inner bark, and debarking enhances the degradation.

Three-dimensional reconstruction of Picea wilsonii Mast. pollen grains using automated electron microscopy.

Three-dimensional electron microscopy (3D-EM) has attracted considerable attention because of its ability to provide detailed information with respect to developmental analysis. However, large-scale high-resolution 3D reconstruction of biological samples remains challenging. Herein, we present a 3D view of a Picea wilsonii Mast. pollen grain with 100 nm axial and 38.57 nm lateral resolution using AutoCUTS-SEM (automatic collector of ultrathin sections-scanning electron microscopy). We established a library of 3,127 100 nm thick serial sections of pollen grains for preservation and observation, demonstrating that the protocol can be used to analyze large-volume samples. After obtaining the SEM images, we reconstructed an entire pollen grain comprising 734 serial sections. The images produced by 3D reconstruction clearly revealed the main components of the P. wilsonii pollen grain, i.e., two sacci and pollen corpus, tube cell, generative cell, and two degenerated prothallial cells, and their internal organization. In addition, we performed a quantitative analysis of the different pollen grain cells, including sacci, and found that there were 202 connections within a saccus SEM image. Thus, for the first time, this study provided a global 3D view of the entire pollen grain, which will be useful for analyzing pollen development and growth.

Tissue-specific transposon-associated small RNAs in the gymnosperm tree, Norway spruce.

BACKGROUND: Small RNAs (sRNAs) are regulatory molecules impacting on gene expression and transposon activity. MicroRNAs (miRNAs) are responsible for tissue-specific and environmentally-induced gene repression. Short interfering RNAs (siRNA) are constitutively involved in transposon silencing across different type of tissues. The male gametophyte in angiosperms has a unique set of sRNAs compared to vegetative tissues, including phased siRNAs from intergenic or genic regions, or epigenetically activated siRNAs. This is contrasted by a lack of knowledge about the sRNA profile of the male gametophyte of gymnosperms. RESULTS: Here, we isolated mature pollen from male cones of Norway spruce and investigated its sRNA profiles. While 21-nt sRNAs is the major size class of sRNAs in needles, in pollen 21-nt and 24-nt sRNAs are the most abundant size classes. Although the 24-nt sRNAs were exclusively derived from TEs in pollen, both 21-nt and 24-nt sRNAs were associated with TEs. We also investigated sRNAs from somatic embryonic callus, which has been reported to contain 24-nt sRNAs. Our data show that the 24-nt sRNA profiles are tissue-specific and differ between pollen and cell culture. CONCLUSION: Our data reveal that gymnosperm pollen, like angiosperm pollen, has a unique sRNA profile, differing from vegetative leaf tissue. Thus, our results reveal that angiosperm and gymnosperm pollen produce new size classes not present in vegetative tissues; while in angiosperm pollen 21-nt sRNAs are generated, in the gymnosperm Norway spruce 24-nt sRNAs are generated. The tissue-specific production of distinct TE-derived sRNAs in angiosperms and gymnosperms provides insights into the diversification process of sRNAs in TE silencing pathways between the two groups of seed plants.

The role of reactive oxygen species in pollen germination in Picea pungens (blue spruce).

KEY MESSAGE: Endogenous ROS, including those produced by NADPH oxidase, are required for spruce pollen germination and regulate membrane potential in pollen tubes; [Formula: see text] and H 2 O 2 are unevenly distributed along the tube. Recently, the key role of reactive oxygen species (ROS) in plant reproduction has been decisively demonstrated for angiosperms. This paper is dedicated to the involvement of ROS in pollen germination of gymnosperms, which remained largely unknown. We found that ROS are secreted from pollen grains of blue spruce during the early stage of activation. The localization of different ROS in pollen tube initials and pollen tubes demonstrated the accumulation of H2O2 in pollen tube apex. Colocalization with mitochondria-derived [Formula: see text] showed that H2O2 is produced in mitochondria and amyloplasts in addition to its apical gradient in the cytosol. The necessity of intracellular ROS and, particularly, [Formula: see text] for pollen germination was demonstrated using different antioxidants. ·OH and extracellular ROS, on the contrary, were found to be not necessary for germination. Exogenous hydrogen peroxide did not affect the germination efficiency but accelerated pollen tube growth in a concentration-dependent manner. The optical measurements of membrane potential showed that in spruce pollen tubes there is a gradient which is controlled by H+-ATPase, potassium- and calcium-permeable channels, anion channels and ROS, as demonstrated by inhibitory analysis. An important role of NADPH oxidase in the regulation of ROS balance in particular, and in germination in general, has been demonstrated by inhibiting the enzyme, which leads to the reduction in ROS release, depolarization of pollen tube plasma membrane, and blocking of pollen germination.

Nonlinear transfer of elements from soil to plants: impact on radioecological modeling.

In radioecology, transfer of radionuclides from soil to plants is typically described by a concentration ratio (CR), which assumes linearity of transfer with soil concentration. Nonlinear uptake is evidenced in many studies, but it is unclear how it should be taken into account in radioecological modeling. In this study, a conventional CR-based linear model, a nonlinear model derived from observed uptake into plants, and a new simple model based on the observation that nonlinear uptake leads to a practically constant concentration in plant tissues are compared. The three models were used to predict transfer of (234)U, (59)Ni and (210)Pb into spruce needles. The predictions of the nonlinear and the new model were essentially similar. In contrast, plant radionuclide concentration was underestimated by the linear model when the total element concentration in soil was relatively low, but within the range commonly observed in nature. It is concluded that the linear modeling could easily be replaced by a new approach that more realistically reflects the true processes involved in the uptake of elements into plants. The new modeling approach does not increase the complexity of modeling in comparison with CR-based linear models, and data needed for model parameters (element concentrations) are widely available.

Allocation of freshly assimilated carbon into primary and secondary metabolites after in situ ¹³C pulse labelling of Norway spruce (Picea abies).

Plants allocate carbon (C) to sink tissues depending on phenological, physiological or environmental factors. We still have little knowledge on C partitioning into various cellular compounds and metabolic pathways at various ecophysiological stages. We used compound-specific stable isotope analysis to investigate C partitioning of freshly assimilated C into tree compartments (needles, branches and stem) as well as into needle water-soluble organic C (WSOC), non-hydrolysable structural organic C (stOC) and individual chemical compound classes (amino acids, hemicellulose sugars, fatty acids and alkanes) of Norway spruce (Picea abies) following in situ (13)C pulse labelling 15 days after bud break. The (13)C allocation within the above-ground tree biomass demonstrated needles as a major C sink, accounting for 86% of the freshly assimilated C 6 h after labelling. In needles, the highest allocation occurred not only into the WSOC pool (44.1% of recovered needle (13)C) but also into stOC (33.9%). Needle growth, however, also caused high (13)C allocation into pathways not involved in the formation of structural compounds: (i) pathways in secondary metabolism, (ii) C-1 metabolism and (iii) amino acid synthesis from photorespiration. These pathways could be identified by a high (13)C enrichment of their key amino acids. In addition, (13)C was strongly allocated into the n-alkyl lipid fraction (0.3% of recovered (13)C), whereby (13)C allocation into cellular and cuticular exceeded that of epicuticular fatty acids. (13)C allocation decreased along the lipid transformation and translocation pathways: the allocation was highest for precursor fatty acids, lower for elongated fatty acids and lowest for the decarbonylated n-alkanes. The combination of (13)C pulse labelling with compound-specific (13)C analysis of key metabolites enabled tracing relevant C allocation pathways under field conditions. Besides the primary metabolism synthesizing structural cell compounds, a complex network of pathways consumed the assimilated (13)C and kept most of the assimilated C in the growing needles.

Wood species affect the degradation of crude oil in beach sand.

The addition of wood chips as a co-substrate can promote the degradation of oil in soil. Therefore, in the present study, the tree species-specific impact of wood chips of Scots pine (Pinus sylvestris L.), Norway spruce (Picea abies L.) and Western balsam poplar (Populus trichocarpa L.) on the degradation of crude oil was tested in beach sand in a 4-week incubation experiment. The CO2-C release increased in the order of control without wood chips < +spruce < +pine < +poplar. Initial and final hydrocarbon concentrations (C10 to C40), as indicators for the oil degradation, were determined with gas chromatography-flame ionization detection (GC-FID). The degradation increased for the light fraction (C10 to C22), the heavy fraction (C23 to C40) as well as the whole range (C10 to C40) in the order of control without wood chips (f(degrad.) = 23% vs. 0% vs. 12%) < +poplar (f(degrad.) = 49% vs. 19% vs. 36%) < +spruce (f(degrad.) = 55% vs. 34% vs. 46%) < +pine (f(degrad.) = 60% vs. 44% vs. 53%), whereas the heavy fraction was less degraded in comparison to the light fraction. It can be concluded, that the tree species-specific wood quality is a significant control of the impact on the degradation of hydrocarbons, and pine wood chips might be promising, possibly caused by their lower decomposability and lower substrate replacement than the other wood species.

Embryogenic potential and expression of embryogenesis-related genes in conifers are affected by treatment with a histone deacetylase inhibitor.

Somatic embryogenesis is used for vegetative propagation of conifers. Embryogenic cultures can be established from zygotic embryos; however, the embryogenic potential decreases during germination. In Arabidopsis, LEAFY COTYLEDON (LEC) genes are expressed during the embryonic stage, and must be repressed to allow germination. Treatment with the histone deacetylase inhibitor trichostatin A (TSA) causes de-repression of LEC genes. ABSCISIC ACID3 (ABI3) and its Zea mays ortholog VIVIPAROUS1 (VP1) act together with the LEC genes to promote embryo maturation. In this study, we have asked the question whether TSA treatment in a conifer affects the embryogenic potential and the expression of embryogenesis-related genes. We isolated two conifer LEC1-type HAP3 genes, HAP3A and HAP3B, from Picea abies and Pinus sylvestris. A comparative phylogenetic analysis of plant HAP3 genes suggests that HAP3A and HAP3B are paralogous genes originating from a duplication event in the conifer lineage. The expression of HAP3A is high, in both somatic and zygotic embryos, during early embryo development, but decreases during late embryogeny. In contrast, the expression of VP1 is initially low but increases during late embryogeny. After exposure to TSA, germinating somatic embryos of P. abies maintain the competence to differentiate embryogenic tissue, and simultaneously the germination progression is partially inhibited. Furthermore, when embryogenic cultures of P. abies are exposed to TSA during embryo maturation, the maturation process is arrested and the expression levels of PaHAP3A and PaVP1 are maintained, suggesting a possible link between chromatin structure and expression of embryogenesis-related genes in conifers.

Does nitrogen deposition increase forest production? The role of phosphorus.

Effects of elevated N deposition on forest aboveground biomass were evaluated using long-term data from N addition experiments and from forest observation plots in Switzerland. N addition experiments with saplings were established both on calcareous and on acidic soils, in 3 plots with Fagus sylvatica and in 4 plots with Picea abies. The treatments were conducted during 15 years and consisted of additions of dry NH4NO3 at rates of 0, 10, 20, 40, 80, and 160 kg N ha(-1) yr(-1). The same tree species were observed in permanent forest observation plots covering the time span between 1984 and 2007, at modeled N deposition rates of 12-46 kg N ha(-1) yr(-1). Experimental N addition resulted in either no change or in a decreased shoot growth and in a reduced phosphorus concentration in the foliage in all experimental plots. In the forest, a decrease of foliar P concentration was observed between 1984 and 2007, resulting in insufficient concentrations in 71% and 67% of the Fagus and Picea plots, respectively, and in an increasing N:P ratio in Fagus. Stem increment decreased during the observation period even if corrected for age. Forest observations suggest an increasing P limitation in Swiss forests especially in Fagus which is accompanied by a growth decrease whereas the N addition experiments support the hypothesis that elevated N deposition is an important cause for this development.

Overexpression of PwTUA1, a pollen-specific tubulin gene, increases pollen tube elongation by altering the distribution of alpha-tubulin and promoting vesicle transport.

Tubulin genes are intimately associated with cell division and cell elongation, which are central to plant secondary cell wall development. However, their roles in pollen tube polar growth remain elusive. Here, a TUA1 gene from Picea wilsonii, which is specifically expressed in pollen, was isolated. Semi-quantitative RT-PCR analysis showed that the amount of PwTUA1 transcript varied at each stage of growth of the pollen tube and was induced by calcium ions and boron. Transient expression analysis in P. wilsonii pollen indicated that PwTUA1 improved pollen germination and pollen tube growth. The pollen of transgenic Arabidopsis overexpressing PwTUA1 also showed a higher percentage of germination and faster growth than wild-type plants not only in optimal germination medium, but also in medium supplemented with elevated levels of exogenous calcium ions or boron. Immunofluorescence and electron microscopy showed alpha-tubulin to be enriched and more vesicles accumulated in the apex region in germinating transgenic Arabidopsis pollen compared with wild-type plants. These results demonstrate that PwTUA1 up-regulated by calcium ions and boron contributes to pollen tube elongation by altering the distribution of alpha-tubulin and regulating the deposition of pollen cell wall components during the process of tube growth. The possible role of PwTUA1 in microtubule dynamics and organization was discussed.

Root proliferation of Norway spruce and Scots pine in response to local magnesium supply in soil.

Nutrient sources in soils are often heterogeneously distributed. Although many studies have examined the root responses to local N and P enrichments in the soil, less research was conducted on root responses to Mg patches. New roots of pre-grown Mg-insufficient and Mg-sufficient plants of Norway spruce (Picea abies [L.] Karst.) and Scots pine (Pinus sylvestris L.) seedlings were allowed to grow into four other pots of equal size, which were placed under the tree-bearing pot. Soils in the lower pots were either unfertilised, or supplied with Mg, or NPK or a mixture of NPKMg sources. Plants were harvested after 9 months of growth. Compared to the corresponding controls (Mg versus unfertilised and NPKMg versus NPK), Mg additions did not have a significant effect on either root dry matter, total root length (TRL) or specific root length (SRL), irrespective of tree species and plant Mg nutritional status. In contrast, NPK and NPKMg additions significantly increased the root dry matter and TRL in the nutrient-rich soil patch, and decreased SRL in Norway spruce. However, the observed root morphological changes did not occur in Scots pine. Root Mg concentrations were increased in Mg-rich soil patches, but those accumulations varied with tree species. Mg accumulation in a marked patch was measured only in newly grown roots of Mg-sufficient Norway spruce, whereas a more homogenous distribution of Mg concentration was observed for all newly grown roots in Mg-insufficient trees in the four soil treatments. In Scots pine, Mg accumulations occurred in both Mg-insufficient and Mg-sufficient plants. These results suggest that Mg patches in the soil may not lead to a local increase in root growth, but to Mg uptake and root Mg accumulation. Tree roots react differently to Mg patches in comparison to their response to N or P patches in the soil.

Combined proteomic and cytological analysis of Ca2+-calmodulin regulation in Picea meyeri pollen tube growth.

Ca2+-calmodulin (Ca2+-CaM) is a critical molecule that mediates cellular functions by interacting with various metabolic and signaling pathways. However, the protein expression patterns and accompanying serial cytological responses in Ca2+-CaM signaling deficiency remain enigmatic. Here, we provide a global analysis of the cytological responses and significant alterations in protein expression profiles after trifluoperazine treatment in Picea meyeri, which abrogates Ca2+-CaM signaling. Ninety-three differentially displayed proteins were identified by comparative proteomics at different development stages and were assigned to different functional categories closely related to tip growth machinery. The inhibition of Ca2+-CaM signaling rapidly induced an increase in extracellular Ca2+ influx, resulting in dramatically increased cytosolic Ca2+ concentrations and ultrastructural abnormalities in organelles as the primary responses. Secondary and tertiary alterations included actin filament depolymerization, disrupted patterns of endocytosis and exocytosis, and cell wall remodeling, ultimately resulting in perturbed pollen tube extension. In parallel with these cytological events, time-course experiments revealed that most differentially expressed proteins showed time-dependent quantitative changes (i.e. some signaling proteins and proteins involved in organelle functions and energy production changed first, followed by alterations in proteins related to cytoskeletal organization, secretory pathways, and polysaccharide synthesis). Taken together, Ca2+-CaM dysfunction induced serial cytological responses and temporal changes in protein expression profiles, indicating the pivotal role of Ca2+-CaM in the regulation of tip growth machinery.

The level of free intracellular zinc mediates programmed cell death/cell survival decisions in plant embryos.

Zinc is a potent regulator of programmed cell death (PCD) in animals. While certain, cell-type-specific concentrations of intracellular free zinc are required to protect cells from death, zinc depletion commits cells to death in diverse systems. As in animals, PCD has a fundamental role in plant biology, but its molecular regulation is poorly understood. In particular, the involvement of zinc in the control of plant PCD remains unknown. Here, we used somatic embryos of Norway spruce (Picea abies) to investigate the role of zinc in developmental PCD, which is crucial for correct embryonic patterning. Staining of the early embryos with zinc-specific molecular probes (Zinquin-ethyl-ester and Dansylaminoethyl-cyclen) has revealed high accumulation of zinc in the proliferating cells of the embryonal masses and abrupt decrease of zinc content in the dying terminally differentiated suspensor cells. Exposure of early embryos to a membrane-permeable zinc chelator N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine led to embryonic lethality, as it induced ectopic cell death affecting embryonal masses. This cell death involved the loss of plasma membrane integrity, metacaspase-like proteolytic activity, and nuclear DNA fragmentation. To verify the anti-cell death effect of zinc, we incubated early embryos with increased concentrations of zinc sulfate. Zinc supplementation inhibited developmental PCD and led to suppression of terminal differentiation and elimination of the embryo suspensors, causing inhibition of embryo maturation. Our data demonstrate that perturbation of zinc homeostasis disrupts the balance between cell proliferation and PCD required for plant embryogenesis. This establishes zinc as an important cue governing cell fate decisions in plants.

[Effects of Fargesia denudata density on its litterfall production, nutrient return, and nutrient use efficiency].

A two-year study on the effects of three densities (D1, 80 +/- 5 stems x m(-2); D2, 140 +/- 8 stems x m(-2); and D3, 220 +/- 11 stems x m(-2)) of Fargesia denudate in a F. denudata--Picea purpurea natural forest on the F. denudate litterfall production, nutrient return, and nutrient use efficiency showed that at the densities of D1, D2 and D3, the annual litterfall production was 793.2, 1135.7 and 1458.5 kg x hm(-2), carbon return was 370.7, 516.2 and 671.5 kg x hm(-2), and the total return of N, P, K, Ca and Mg was 16.3, 22.9 and 29.3 kg x hm(-2), respectively. Ca had the highest return (45%-48% of the total), followed by N (24%-29%), and the lowest were P and Mg (3%-5%), with the sequence of Ca > N > K > Mg and P. The peaks of litterfall production and nutrients return at the three densities were appeared in October (by the end of F. denudate growth season), and another peak at D3 was observed in August. There were no significant differences in the re-allocation of N and K in F. denudate leaves at the three densities, but the P re-allocation and the Ca and Mg accumulation increased with increasing density. The litterfall P use efficiency was the highest and increased with increasing density, implying that P could be a limiting factor for the growth and regeneration of F. denudate.

Seasonal profiles of sulphur, phosphorus, and potassium in Norway spruce wood.

Seasonal profiles of sulphur, phosphorus, and potassium content in the wood of trees have been established for the first time. This became possible by using a novel laser ablation system coupled to HR-ICP-MS for measuring these elements in Norway spruce drill cores. This technique combines excellent spatial resolution with superior detection power, and makes it possible to measure low element concentrations even in relatively narrow annual rings. Despite its low quantity in wood, sulphur is an important macronutrient for plants and seems to display seasonal variations of its concentration, which correspond to actual theories of sulphur metabolism in plants. A similar seasonal pattern was also found for phosphorus, another crucial element in tree nutrition. This was unexpected, because it was previously assumed that the distribution of phosphorus remains constant throughout the year. Potassium, the third element measured, seems to be especially accumulated in the latewood. The profiles presented in this article suggest a seasonal variation, revealing some new aspects of Norway spruce (PICEA ABIES) metabolism.

Roles of the ubiquitin/proteasome pathway in pollen tube growth with emphasis on MG132-induced alterations in ultrastructure, cytoskeleton, and cell wall components.

The ubiquitin/proteasome pathway represents one of the most important proteolytic systems in eukaryotes and has been proposed as being involved in pollen tube growth, but the mechanism of this involvement is still unclear. Here, we report that proteasome inhibitors MG132 and epoxomicin significantly prevented Picea wilsonii pollen tube development and markedly altered tube morphology in a dose- and time-dependent manner, while hardly similar effects were detected when cysteine-protease inhibitor E-64 was used. Fluorogenic kinetic assays using fluorogenic substrate sLLVY-AMC confirmed MG132-induced inhibition of proteasome activity. The inhibitor-induced accumulation of ubiquitinated proteins (UbPs) was also observed using immunoblotting. Transmission electron microscopy revealed that MG132 induces endoplasmic reticulum (ER)-derived cytoplasmic vacuolization. Immunogold-labeling analysis demonstrated a significant accumulation of UbPs in degraded cytosol and dilated ER in MG132-treated pollen tubes. Fluorescence labeling with fluorescein isothiocyanate-phalloidin and beta-tubulin antibody revealed that MG132 disrupts the organization of F-actin and microtubules and consequently affects cytoplasmic streaming in pollen tubes. However, tip-focused Ca2+ gradient, albeit reduced, seemingly persists after MG132 treatment. Finally, fluorescence labeling with antipectin antibodies and calcofluor indicated that MG132 treatment induces a sharp decline in pectins and cellulose. This result was confirmed by Fourier transform infrared analysis, thus demonstrating for the first time the inhibitor-induced weakening of tube walls. Taken together, these findings suggest that MG132 treatment promotes the accumulation of UbPs in pollen tubes, which induces ER-derived cytoplasmic vacuolization and depolymerization of cytoskeleton and consequently strongly affects the deposition of cell wall components, providing a mechanistic framework for the functions of proteasome in the tip growth of pollen tubes.

Differential display proteomic analysis of Picea meyeri pollen germination and pollen-tube growth after inhibition of actin polymerization by latrunculin B.

To investigate roles of the actin cytoskeleton in growth of the pollen tube of Picea meyeri, we used the actin polymerization inhibitor latrunculin B (LATB) under quantitatively controlled conditions. At low concentrations, LATB inhibited polymerization of the actin cytoskeleton in the growing pollen tube, which rapidly inhibited tip growth. The proteomic approach was used to analyse protein expression-profile changes during pollen germination and subsequent pollen-tube development with disturbed organization of the actin cytoskeleton. Two-dimensional electrophoresis and staining with Coomassie Brilliant Blue revealed nearly 600 protein spots. A total of 84 of these were differentially displayed at different hours with varying doses of LATB, and 53 upregulated or downregulated proteins were identified by mass spectrometry. These proteins were grouped into distinct functional categories including signalling, actin cytoskeleton organization, cell expansion and carbohydrate metabolism. Moreover, actin disruption affected the morphology of Golgi stacks, mitochondria and amyloplasts, along with a differential expression of proteins involved in their functions. These findings provide new insights into the multifaceted mechanism of actin cytoskeleton functions and its interaction with signalling, cell-expansion machinery and energy-providing pathways.

Calcium gradients in conifer pollen tubes; dynamic properties differ from those seen in angiosperms.

Pollen tubes are an established model system for examining polarized cell growth. The focus here is on pollen tubes of the conifer Norway spruce (Picea abies, Pinaceae); examining the relationship between cytosolic free Ca2+, tip elongation, and intracellular motility. Conifer pollen tubes show important differences from their angiosperm counterparts; they grow more slowly and their organelles move in an unusual fountain pattern, as opposed to reverse fountain, in the tip. Ratiometric ion imaging of growing pollen tubes, microinjected with fura-2-dextran, reveals a tip-focused [Ca2+]i gradient extending from 450 nM at the extreme apex to 225 nM at the base of the tip clear zone. Injection of 5,5' dibromo-BAPTA does not dissipate the apical gradient, but stops cell elongation and uniquely causes rapid, transient increases of apical free Ca2+. The [Ca2+]i gradient is, however, dissipated by reversible perfusion of extracellular caffeine. When the basal cytosolic free Ca2+ concentration falls below 150 nM, again a large increase in apical [Ca2+]i occurs. An external source of calcium is not required for germination but significantly enhances elongation. However, both germination and elongation are significantly inhibited by the inclusion of calcium channels blockers, including lanthanum, gadolinium, or verapamil. Modulation of intracellular calcium also affects organelle position and motility. Extracellular perfusion of lanthanides reversibly depletes the apical [Ca2+]i gradient, altering organelle positioning in the tip. Later, during recovery from lanthanide perfusion, organelle motility switches direction to a reverse fountain. When taken together these data show a unique interplay in Picea abies pollen tubes between intracellular calcium and the motile processes controlling cellular organization.

An improved MUG fluorescent assay for the determination of GUS activity within transgenic tissue of woody plants.

Despite the success of the MUG fluorometric assay for quantitative analysis of beta-glucuronidase (GUS) activity within a vast array of transgenic plant species and tissues, attempts to apply this protocol for analysis of woody plants has been found to be problematic, primarily due to the interfering effects of phenolics and other secondary metabolites. Our analysis of transgenic spruce needles and poplar leaves illustrates that low tissue mass to extract volume, along with the inclusion of polyvinylpolypyrolidone and either beta-mercaptoethanol or metabisulphite, are essential for producing reliable results. The primary action of these additives was found to involve increased GUS extractability and the preservation of GUS activity during extract manipulation, but they were not completely effective in eliminating GUS enzymatic inhibitors. Normalization of GUS activity upon DNA concentration was also found to be an effective alternative to protein concentration, providing the ability to make cross-species and inter-tissue comparisons of gusA transgene activity.