Transcriptome responses to temperature, water availability and photoperiod are conserved among mature trees of two divergent Douglas-fir provenances from a coastal and an interior habitat.

BACKGROUND: Local adaptation and phenotypic plasticity are important components of plant responses to variations in environmental conditions. While local adaptation has been widely studied in trees, little is known about plasticity of gene expression in adult trees in response to ever changing environmental conditions in natural habitats. Here we investigate plasticity of gene expression in needle tissue between two Douglas-fir provenances represented by 25 adult trees using deep RNA sequencing (RNA-Seq). RESULTS: Using linear mixed models we investigated the effect of temperature, soil water availability and photoperiod on the abundance of 59189 detected transcripts. Expression of more than 80 % of all identified transcripts revealed a response to variations in environmental conditions in the field. GO term overrepresentation analysis revealed gene expression responses to temperature, soil water availability and photoperiod that are highly conserved among many plant taxa. However, expression differences between the two Douglas-fir provenances were rather small compared to the expression differences observed between individual trees. Although the effect of environment on global transcript expression was high, the observed genotype by environment (GxE) interaction of gene expression was surprisingly low, since only 21 of all detected transcripts showed a GxE interaction. CONCLUSIONS: The majority of the transcriptome responses in plant leaf tissue is driven by variations in environmental conditions. The small variation between individuals and populations suggests strong conservation of this response within Douglas-fir. Therefore we conclude that plastic transcriptome responses to variations in environmental conditions are only weakly affected by local adaptation in Douglas-fir.

Bisphenol A disrupts microtubules and induces multipolar spindles in dividing root tip cells of the gymnosperm Abies cephalonica.

The effects of bisphenol A (BPA), an endocrine chemical disruptor extensively used in the plastic and epoxy resin industry, on dividing root tip cells of the gymnosperm Abies cephalonica Loudon were investigated by confocal laser scanning microscopy after tubulin and endoplasmic reticulum immunolocalization and DNA staining. Microtubule arrays of all mitotic stages were disrupted within a few hours of treatment: preprophase bands exhibited asymmetric width; prometaphase, metaphase and anaphase spindles appeared sharply pointed, sigmoid or multipolar; phragmoplast microtubules were elongated and occasionally bended toward the daughter nuclei. Depending on the mitotic stage, the chromosomes appeared condensed at prophase, as a compact mass at metaphase and anaphase, unsegregated or bridged at telophase. Endoplasmic reticulum patterns were also affected, reflecting those of the respective microtubule arrays. Recovery of the microtubules after oryzalin treatment was more effective in a BPA solution than in water. It is concluded that the plant mitotic apparatus microtubules are very sensitive to BPA, the effect of which depends on the specific cell cycle stage. The formation of multipolar spindles is reminiscent of animal cells and is ascribed to the induction of multiple microtubule nucleation sites, deriving from the centrosomal properties of gymnosperms.

Modelling the impact of climate change and atmospheric N deposition on French forests biodiversity.

A dynamic coupled biogeochemical-ecological model was used to simulate the effects of nitrogen deposition and climate change on plant communities at three forest sites in France. The three sites had different forest covers (sessile oak, Norway spruce and silver fir), three nitrogen loads ranging from relatively low to high, different climatic regions and different soil types. Both the availability of vegetation time series and the environmental niches of the understory species allowed to evaluate the model for predicting the composition of the three plant communities. The calibration of the environmental niches was successful, with a model performance consistently reasonably high throughout the three sites. The model simulations of two climatic and two deposition scenarios showed that climate change may entirely compromise the eventual recovery from eutrophication of the simulated plant communities in response to the reductions in nitrogen deposition. The interplay between climate and deposition was strongly governed by site characteristics and histories in the long term, while forest management remained the main driver of change in the short term.

Are Early Somatic Embryos of the Norway Spruce (Picea abies (L.) Karst.) Organised?

BACKGROUND: Somatic embryogenesis in conifer species has great potential for the forestry industry. Hence, a number of methods have been developed for their efficient and rapid propagation through somatic embryogenesis. Although information is available regarding the previous process-mediated generation of embryogenic cells to form somatic embryos, there is a dearth of information in the literature on the detailed structure of these clusters. METHODOLOGY/PRINCIPAL FINDINGS: The main aim of this study was to provide a more detailed structure of the embryogenic tissue clusters obtained through the in vitro propagation of the Norway spruce (Picea abies (L.) Karst.). We primarily focused on the growth of early somatic embryos (ESEs). The data on ESE growth suggested that there may be clear distinctions between their inner and outer regions. Therefore, we selected ESEs collected on the 56th day after sub-cultivation to dissect the homogeneity of the ESE clusters. Two colourimetric assays (acetocarmine and fluorescein diacetate/propidium iodide staining) and one metabolic assay based on the use of 2,3,5-triphenyltetrazolium chloride uncovered large differences in the metabolic activity inside the cluster. Next, we performed nuclear magnetic resonance measurements. The ESE cluster seemed to be compactly aggregated during the first four weeks of cultivation; thereafter, the difference between the 1H nuclei concentration in the inner and outer clusters was more evident. There were clear differences in the visual appearance of embryos from the outer and inner regions. Finally, a cluster was divided into six parts (three each from the inner and the outer regions of the embryo) to determine their growth and viability. The innermost embryos (centripetally towards the cluster centre) could grow after sub-cultivation but exhibited the slowest rate and required the longest time to reach the common growth rate. To confirm our hypothesis on the organisation of the ESE cluster, we investigated the effect of cluster orientation on the cultivation medium and the influence of the change of the cluster's three-dimensional orientation on its development. Maintaining the same position when transferring ESEs into new cultivation medium seemed to be necessary because changes in the orientation significantly affected ESE growth. CONCLUSIONS AND SIGNIFICANCE: This work illustrated the possible inner organisation of ESEs. The outer layer of ESEs is formed by individual somatic embryos with high metabolic activity (and with high demands for nutrients, oxygen and water), while an embryonal group is directed outside of the ESE cluster. Somatic embryos with depressed metabolic activity were localised in the inner regions, where these embryonic tissues probably have a very important transport function.

Combined effect of elevated UVB, elevated temperature and fertilization on growth, needle structure and phytochemistry of young Norway spruce (Picea abies) seedlings.

Simultaneously with warming climate, other climatic and environmental factors are also changing. Here, we investigated for the first time the effects of elevated temperature, increased ultraviolet-B (UVB) radiation, fertilization and all combinations of these on the growth, secondary chemistry and needle structure of 1-year-old Norway spruce (Picea abies (L.) Karst.) seedlings in an outdoor experiment. After one growing season, elevated temperature increased root : shoot ratio and concentrations of needle piperidine alkaloids, while concentrations of needle catechins and acetophenones and bark flavonoids decreased compared with ambient temperature seedlings. UVB-radiation increased concentrations of bark condensed tannins, while fertilization increased total biomass and concentrations of needle catechins. In addition to the main effects, concentrations of some individual phenolic compounds showed UV × temperature or UV × temperature × fertilization interactions, and fertilization modified temperature response on root : shoot ratio. All the treatments described here affected the defence chemistry profiles of the seedlings, which may imply some changes in plant-herbivore interactions in connection with changing climate. The interactions between treatments indicate a need for further experiments involving several simultaneously affecting environmental changes.

Changes in ATP, glucose-6-phosphate and NAD(P)H cellular levels during the proliferation and maturation phases of Abies alba Mill. embryogenic cultures.

The aim of the present study was to evaluate the adenosine triphospate (ATP), glucose-6-phosphate (glu-6P) and reduced form of nicotinamide adenine dinucleotide phosphate (NAD(P)H) cellular levels during the proliferation and maturation phases of Abies alba Mill. somatic embryos. For a better understanding of the dynamics of these parameters during the proliferation cycle, four embryonic cell lines were tested. During the maturation period, three independent experiments were conducted, focused on the effects of PEG-4000 (5 or 10% (w/v)) and abscisic acid (16, 32 or 64 µM) applied together (Experiments A and B) or with addition of gibberellic acid (Experiment C) on the dynamics of bio-energetic molecules and on the mean number of cotyledonary somatic embryos. Our results demonstrated that the cellular levels of bio-energetic molecules strongly depended on the composition of maturation media. Generally, the higher the number of cotyledonary embryos produced, the higher the level of ATP observed after a 2-week maturation period. The cellular level of ATP, glu-6P and NAD(P)H increased, particularly after the transition from the proliferation to the maturation phase when the differentiation and growth of somatic embryos occurred.

Fulvic acid affects proliferation and maturation phases in Abies cephalonica embryogenic cells.

Embryogenic cell masses (ECM) of Abies cephalonica were grown on proliferation media in the presence and absence of fulvic acid (FA), whose molecular composition and conformational rigidity were evaluated by CPMAS-¹³C NMR spectroscopy. To assess the physiological effects of this humic material during proliferation and maturation stages of somatic embryogenesis (SE), proliferation rate, proportion of consecutive developmental stages of pro-embryogenic masses (PEM), cellular ATP and glucose-6-phosphate were evaluated at regular intervals. FA increased the proliferation rate, especially during the early sampling days, and the percentage of PEM in their advanced developmental stage. Cellular ATP and glucose-6-phospahte were increased by FA pre-treatment during the maturation phase. Furthermore, the effects of the anti-auxin p-chlorophenoxyisobutyric acid (PCIB), such as a decrease of growth and the enhancement of PEM III induction, were inverted by FA. Proton pumping ATPase and PPase activities were decreased in microsomes from PCIB-treated ECM, while they increased in the presence of FA. This fulvic matter also induced a delay in somatic embryo formation during the maturation phase. Both the improvement of the PEM proliferation and the reduction of the subsequent maturation process of A. cephalonica are explained by a release from the complex humic structure of low molecular-weight molecules, which may interact with the plant hormonal signaling pathway. These effects appear to be related to the hydrophilic and conformationally labile nature of FA. The structure-activity relationship observed here suggests that the influence of FA on ECM may be attributed to specific bioactive molecules that are preferentially released from the FA loose superstructure.

Mitochondrial bioenergetics linked to the manifestation of programmed cell death during somatic embryogenesis of Abies alba.

The present work reports changes in bioenergetic parameters and mitochondrial activities during the manifestation of two events of programmed cell death (PCD), linked to Abies alba somatic embryogenesis. PCD, evidenced by in situ nuclear DNA fragmentation (TUNEL assay), DNA laddering and cytochrome c release, was decreased in maturing embryogenic tissue with respect to the proliferation stage. In addition, the major cellular energetic metabolites (ATP, NAD(P)H and glucose-6-phosphate) were highered during maturation. The main mitochondrial activities changed during two developmental stages. Mitochondria, isolated from maturing, with respect to proliferating cell masses, showed an increased activity of the alternative oxidase, external NADH dehydrogenase and fatty-acid mediated uncoupling. Conversely, a significant decrease of the mitochondrial K (ATP)(+) channel activity was observed. These results suggest a correlation between mitochondrial activities and the manifestation of PCD during the development of somatic embryos. In particular, it is suggested that the K (ATP)(+) channel activity could induce an entry of K(+) into the matrix, followed by swelling and a release of cytochrome c during proliferation, whereas the alternative pathways, acting as anti-apoptotic factors, may partially counteract PCD events occurring during maturation of somatic embryos.

Image analysis of epicuticular damage to foliage caused by dry deposition of the air pollutant nitric acid.

Nitric acid vapor is produced by the same photochemical processes that produce ozone. In the laboratory, concentrated nitric acid is a strong acid and a powerful oxidant. In the environment, where the concentrations are much lower, it is an innocuous source of plant nitrogen. As an air pollutant, which mode of action does dry deposition of nitric acid follow? We investigated the effects of dry deposition of nitric acid on the foliage of four tree species native to the western United States. A novel controlled environment, fumigation system enabled a four-week exposure at concentrations consistent with ambient diurnal patterns. Scanning electron microscopy and automated image analysis revealed changes in the epicuticular wax layer during fumigation. Exposure to nitric acid resulted in a reproducible suite of damage symptoms that increased with increasing dose. Each tree species tested exhibited a unique set of damage features, including cracks, lesions, and conformation changes to epicuticular crystallite structures. Dry deposition of atmospheric nitric acid caused substantial perturbation to the epicuticular surface of all four tree species investigated, consistent with the chemical oxidation of epicuticular waxes. Automated image analysis eliminated many biases that can trouble microscopy studies. Trade names and commercial enterprises or products are mentioned solely for information. No endorsements by the U.S. Department of Agriculture are implied.

Growth and wood/bark properties of Abies faxoniana seedlings as affected by elevated CO2.

Growth and wood and bark properties of Abies faxoniana seedlings after one year's exposure to elevated CO2 concentration (ambient + 350 (+/- 25) micromol/mol) under two planting densities (28 or 84 plants/m(2)) were investigated in closed-top chambers. Tree height, stem diameter and cross-sectional area, and total biomass were enhanced under elevated CO2 concentration, and reduced under high planting density. Most traits of stem bark were improved under elevated CO2 concentration and reduced under high planting density. Stem wood production was significantly increased in volume under elevated CO2 concentration under both densities, and the stem wood density decreased under elevated CO2 concentration and increased under high planting density. These results suggest that the response of stem wood and bark to elevated CO2 concentration is density dependent. This may be of great importance in a future CO2 enriched world in natural forests where plant density varies considerably. The results also show that the bark/wood ratio in diameter, stem cross-sectional area and dry weight are not proportionally affected by elevated CO2 concentration under the two contrasting planting densities. This indicates that the response magnitude of stem bark and stem wood to elevated CO2 concentration are different but their response directions are the same.

A review of ozone-induced effects on the forests of central Mexico.

The first report on oxidant-induced plant damage in the Valley of Mexico was presented over 30 years ago. Ozone is known to occur in the Mexico City Metropolitan Area and elsewhere as the cause of chlorotic mottling on pine needles that are 2 years old or older as observed in 1976 on Pinus hartwegii and Pinus leiophylla. Visible evidences for the negative effects of ozone on the vegetation of central Mexico include foliar injury expressed as chlorotic mottling and premature defoliation on pines, a general decline of sacred fir, visible symptoms on native forest broadleaved species (e.g. Mexican black cherry). Recent investigations have also indicated that indirect effects are occurring such as limited root colonization by symbiotic fungi on ozone-damaged P. hartwegii trees and a negative influence of the pollutant on the natural regeneration of this species. The negative ozone-induced effects on the vegetation will most likely continue to increase.

Response of antioxidative enzymes of Abies firma seedlings under chronic acid fog exposure and/or drought treatment.

To understand the oxidative stress conditions of momi fir (Abies firma Sieb. et Zucc.) seedlings under acid fog exposure and/or drought stress, we assayed the activities of antioxidative enzymes, such as superoxide dismutase (SOD), catalase (CAT), and ascorbate peroxidase (APX), in the seedlings. We found that SOD and APX activities were not affected by the stress conditions, but drought stress stimulated CAT activity irrespective of acid fog exposure. This suggests the production of a large amount of hydrogen peroxide, which is one of the substances involved in the signal transduction pathway for stomatal closure. However, we previously observed sufficient stomatal closure under drought stress and insufficient stomatal closure under combined acid fog exposure and drought stress. This inconsistency indicates that acid fog exposure affects the signal transduction pathway from the production of hydrogen peroxide to stomatal closure in momi fir seedlings.

Genetic effects of air pollution on forest tree species of the Carpathian Mountains.

The effects of air pollution on the genetic structure of Norway spruce, European silver fir and European beech were studied at four polluted sites in Slovakia, Romania and Czech Republic. In order to reduce potential effects of site heterogeneity on the health condition, pair-wise sampling of pollution-tolerant and sensitive trees was applied. Genotypes of sampled trees were determined at 21 isozyme gene loci of spruce, 18 loci of fir and 15 loci of beech. In comparison with Norway spruce, fewer genetic differences were revealed in beech and almost no differentiation between pollution-tolerant and sensitive trees was observed in fir. In adult stands of Norway spruce, sensitive trees exhibited higher genetic multiplicity and diversity. The decline of pollution-sensitive trees may result thus in a gradual genetic depletion of pollution-exposed populations of Norway spruce through the loss of less frequent alleles with potential adaptive significance to altered stressing regimes in the future. Comparison of the subsets of sensitive and tolerant Norway spruce individuals as determined by presence or absence of discolorations ("spruce yellowing") revealed different heterozygosity at 3 out of 11 polymorphic loci.