Multimodal imaging analysis in silver fir reveals coordination in cellulose and lignin deposition.

Despite lignin being a key component of wood, the dynamics of tracheid lignification are generally overlooked in xylogenesis studies, which hampers our understanding of environmental drivers and blurs the interpretation of isotopic and anatomical signals stored in tree rings. Here, we analyzed cell wall formation in silver fir (Abies alba Mill.) tracheids to determine if cell wall lignification lags behind secondary wall deposition. For this purpose, we applied a multimodal imaging approach combining transmitted light microscopy (TLM), confocal laser scanning microscopy (CLSM), and confocal Raman microspectroscopy (RMS) on anatomical sections of wood microcores collected in northeast France on 11 dates during the 2010 growing season. Wood autofluorescence after laser excitation at 405 and 488 nm associated with the RMS scattering of lignin and cellulose, respectively, which allowed identification of lignifying cells (cells showing lignified and nonlignified wall fractions at the same time) in CLSM images. The number of lignifying cells in CLSM images mirrored the number of wall-thickening birefringent cells in polarized TLM images, revealing highly synchronized kinetics for wall thickening and lignification (similar timings and durations at the cell level). CLSM images and RMS chemical maps revealed a substantial incorporation of lignin into the wall at early stages of secondary wall deposition. Our results show that most of the cellulose and lignin contained in the cell wall undergo concurrent periods of deposition. This suggests a strong synchronization between cellulose and lignin-related features in conifer tree-ring records, as they originated over highly overlapped time frames.

Climate signals in tree-ring δ18 O and δ13 C from southeastern Tibet: insights from observations and forward modelling of intra- to interdecadal variability.

Stable isotopes in tree rings are increasingly used as proxies for climatic and ecophysiological changes. However, uncertainties remain about the strength and consistency of their response to environmental variation at different temporal (i.e. seasonal to inter-decadal) scales. We developed 5 yr of intra-seasonal and 62 yr of early- and late-wood δ13 C and δ18 O series of Smith fir (Abies georgei var. smithii) on the southeastern Tibetan Plateau, and used a process-based forward model to examine the relative importance of environmental and physiological controls on the isotopic data. In this temperate high-altitude region, the response, both δ18 O and δ13 C, is primarily to variations in relative humidity, but by different processes. In δ18 O, the response is via source water δ18 O but also arises from leaf water 18 O enrichment. In δ13 C, the response is via changes in stomatal conductance but is modified by carry-over effects from prior periods. We conclude that tree-ring δ18 O may be a more robust climate proxy than δ13 C, and δ13 C may be more suited to studies of site-related physiological responses to the local environment.

Early-successional ectomycorrhizal fungi effectively support extracellular enzyme activities and seedling nitrogen accumulation in mature forests.

After stand-replacing disturbance, regenerating conifer seedlings become colonized by different ectomycorrhizal fungi (EMF) than the locally adapted EMF communities present on seedlings in mature forests. We studied whether EMF species that colonized subalpine fir (Abies lasiocarpa) seedlings in clearcuts differed from those that colonized seedlings in adjacent mature forests with respect to mycorrhizoplane extracellular enzyme activities (EEAs) and N status of the seedlings. We tested two alternate hypotheses: (1) that EEAs would differ between the two EMF communities, with higher activities associated with forest-origin communities, and (2) that acclimation to soil environment was considerable enough that EEAs would be determined primarily by the soil type in which the ectomycorrhizas were growing. Naturally colonized fir seedlings were reciprocally transplanted between clearcuts and forests, carrying different EMF communities with them. EEAs were influenced more by destination environment than by EMF community. EEAs were as high in early-successional as in late-successional communities in both destination environments. Buds of clearcut-origin seedlings had the same or higher N contents as forest seedlings after a growing season in either environment. These results indicate that (i) symbiotic EMF and/or their associated microbial communities demonstrate substantial ability to acclimate to new field environments; (ii) the ability to produce organic matter-degrading enzymes is not a trait that necessarily distinguishes early- and late-successional EMF communities in symbiosis; (iii) early-successional EMF are as capable of supporting seedling N accumulation in forest soils as late-successional EMF; and (iv) disturbed ecosystems where early-successional EMF are present should have high resilience for organic matter degradation.

Heavy metal concentrations in timberline trees of eastern Tibetan Plateau.

Concentrations of 14 heavy metals (Ag, As, Cd, Co, Cr, Cu, Hg, Mo, Ni, Pb, Sb, Tl, V, and Zn) in needles, twigs, bark and xylem of spruce and fir collected at the timberline of eight sites along the Hengduan Mountains, eastern Tibetan Plateau, are reported. Twigs had the highest concentration for most of elements, while xylem had the lowest concentration. The connections between elements in twigs were much richer than other organ/tissues. Pb, Ni, As, Sb, Co, Cd, Hg, Cr and Tl which are partly through anthropogenic sources and brought in by monsoon, have been accumulated in twigs and needles by wet or dry deposition in south and east sites where are within or near pollutant sources. Under moderate pollution situation, vegetation are able to adjust the nutrient element (Cu and Zn) cycle rate, thus maintain a stable concentration level. Seldom V, Ag, and Mo are from external anthropogenic sources. Needles and twigs can be used as biomonitors for ecosystem environment when needles can simply distinguish the origin of elements and twigs are more sensitive to extra heavy metal input.

Structure of KOH-soluble polysaccharides from сoniferous greens of Norway spruce (Picea abies): The pectin-xylan-AGPs complex. Part 2.

Polymers containing arabinoglucuronoxylan, fucogalactoxyglucan, pectin and arabinogalactan proteins were obtained from PAK isolated from Norway spruce with 7 % KOH. The pectin core of PAK-I2-F-1 and PAK-I2-F-2 was dominated by RG-I, as treatment with 1,4-α-D-polygalacturonase resulted in almost complete removal of homogalacturonan. Interestingly, the above has not affected the co-fractionation of arabinoglucuronoxylan (AGX), arabinogalactan proteins and rhamnogalacturonan I (RG-I). Since pectin was mainly represented by RG-I, we concluded that xylan is specifically associated with RG-I. Correlations in the HMBC spectrum demonstrate intermolecular interactions between the α-L-Rhap (RG-I) and the Xyl (xylan), indicating a covalently bound AGX:RG-I complex via the Xyl-(1→4)-Rha bond: …→2)-[(2,4-ß-D-Xylp)-(1→4)]-[(α-D-GalpA-(1→2)]-α-L-Rhap-(1→4)-α-D-GalpA-(1→…. In PAK-H1-1-F-1 and PAK-H1-1-F-2, parts of RG-I and xylan were removed by enzymolysis. Part of the xylan was probably attached to the above-mentioned RG-I blocks. The removal of part of RG-I, xylan and the disappearance of the signal in the HMBC spectrum indicating the bond between RG-I and xylan confirms that part of the arabinoglucuronoxylan is covalently bound to RG-I. The observed glycosidic linkage contradicts the dominant PCW model in which pectin and hemicellulose polysaccharide networks are considered as independent components. It can be concluded that alkali-soluble xylan from Norway spruce was detected both in the free state and covalently bound to pectin.

Structure of KOH-soluble polysaccharides from сoniferous greens of Norway spruce (Picea abies): The pectin-xylan-AGPs complex. Part 1.

Using 7 % KOH, the polysaccharide PAK has been isolated from the coniferous greens of Norway spruce. PAK was found to contain predominantly arabinoglucuronoxylan, xyloglucan and arabinan, but also pectic polysaccharides, glucomannan and arabinogalactan proteins (AGPs), as determined by 1D/2D NMR analysis. It was found that fractionation of PAK on DEAE-cellulose resulted in simultaneous elution of pectins, arabinoglucuronoxylans and AGPs. It was evident that the content of 4-OMe-α-D-GlcpA and xylose, 1,4-ß-D-GlcpA, and T-ß-D-GlcpA increased with an increase in NaCl concentration. However, 1,4-α-D-GalpA content was almost independent of NaCl concentration, indicating unchanged pectic polysaccharide concentration. Interestingly, pectins extracted with 0.1-0.3 M NaCl solutions were richer in rhamnogalacturonan-I (RG-I) than those extracted with water and 0.01 M NaCl. Conclusion: The content of RG-I, AGPs and arabinoglucuronoxylan rises with rising NaCl concentration. An intense signal indicating an intermolecular linkage between the xylan and RG-I domains, i.e. that part of the arabinoglucuronoxylan is covalently bound to RG-I, is observed in the HMBC spectra of the polysaccharides obtained. The discovery here of a new relationship between rhamnogalacturonan I and xylan contradicts the prevailing cell wall model.

Generalized additive models reveal the intrinsic complexity of wood formation dynamics.

The intra-annual dynamics of wood formation, which involves the passage of newly produced cells through three successive differentiation phases (division, enlargement, and wall thickening) to reach the final functional mature state, has traditionally been described in conifers as three delayed bell-shaped curves followed by an S-shaped curve. Here the classical view represented by the 'Gompertz function (GF) approach' was challenged using two novel approaches based on parametric generalized linear models (GLMs) and 'data-driven' generalized additive models (GAMs). These three approaches (GFs, GLMs, and GAMs) were used to describe seasonal changes in cell numbers in each of the xylem differentiation phases and to calculate the timing of cell development in three conifer species [Picea abies (L.), Pinus sylvestris L., and Abies alba Mill.]. GAMs outperformed GFs and GLMs in describing intra-annual wood formation dynamics, showing two left-skewed bell-shaped curves for division and enlargement, and a right-skewed bimodal curve for thickening. Cell residence times progressively decreased through the season for enlargement, whilst increasing late but rapidly for thickening. These patterns match changes in cell anatomical features within a tree ring, which allows the separation of earlywood and latewood into two distinct cell populations. A novel statistical approach is presented which renews our understanding of xylogenesis, a dynamic biological process in which the rate of cell production interplays with cell residence times in each developmental phase to create complex seasonal patterns.

Cadmium (II) ions removal from aqueous solutions Using Romanian untreated fir tree sawdust a green biosorbent.

Biosorption of cadmium ions from synthetic aqueous solution using popular Romanian fir tree sawdust (Abies Alba) as biosorbent, was investigated in this work. Prior to its utilization the considered biomass was washed, dried and sieved without further chemical treatments. The biosorbent was characterized using humidity, density and elemental analysis determinations and FTIR. FTIR analysis indicated that, on the biomass surface hydroxyl and carboxyl groups are presented. The effect of different biosorption parameters was studied. Higher biomass quantity, neutral pH, slightly elevated temperature and high cadmium ions concentration are all favouring the biosorption process. Equilibrium (Langmuir and Freundlich isotherm), kinetics and thermodynamics of the considered biosorption process were discussed in details. Equilibrium was best described by the Langmuir isotherm, while the kinetic of the process was best described by the pseudo-second-order model, suggesting monolayer coverage and a chemisorption process. Thermodynamic parameters showed that cadmium biosorption process on fir tree sawdust is an endothermic process.

Mode of coniferous wood decay by the white rot fungus Phanerochaete carnosa as elucidated by FTIR and ToF-SIMS.

The softwood degrading white-rot fungus, Phanerochaete carnosa, was investigated for its ability to degrade two coniferous woods: balsam fir and lodgepole pine. P. carnosa grew similarly on these wood species, and like the hardwood-degrading white-rot fungus Ceriporiopsis subvermispora, P. carnosa demonstrated selective degradation of lignin, as observed by Fourier transform infrared spectroscopy and time-of-flight secondary ion mass spectrometry (ToF-SIMS). Lignin degradation across cell walls of decayed pine samples was also evaluated by ToF-SIMS and was shown to be uniform. This study illustrates softwood lignin utilization by a white-rot fungus and reveals the industrial potential of the lignocellulolytic activity elicited by this fungus.

Abies spectabilis-Mediated Silver Nanoparticles Inhibits Cell Growth and Promotes Apoptosis in Breast Cancer MCF-7 Cells.

Breast cancer is the second most cause of mortality among women worldwide due to the uncontrolled proliferation of tumor cells in the mammary epithelial tissues. The silver nanoparticles were formulated from the Abies spectabilis leaf (AS-AgNPs) and characterized by various practices like UV-vis spectroscopy, FTIR, SEM, and XRD. The in vitro anticancer potential of fabricated AS-AgNPs against the MCF-7 cells were analyzed. The MTT test was executed to investigate the cytotoxic nature of fabricated AS-AgNPs against MCF-7 cells. The magnitudes of ROS accumulation and MMP level in the AS-AgNPs supplemented MCF-7 cells were studied using fluorescent staining techniques. Caspase activities were studied using assay kits. The contents of oxidative stress and antioxidant biomarker (TBARS, SOD, CAT, and GSH) levels were scrutinized by standard methods. The expressions of apoptotic markers like Bax and Bcl-2 in the AS-AgNPs administered MCF-7 cells were detected by RT-PCR assay. The MTT findings showed that both extract and fabricated AS-AgNPs remarkably decreased the MCF-7 cells. Nonetheless, both plant extract and AS-AgNPs did not affect the cell viability of MCF-10A cells. Furthermore, the fabricated AS-AgNPs improved the ROS accumulation, and depleted the MMP status in the MCF-7 cells. AS-AgNPs administered MCF-7 cells demonstrated the improved TBARS content and depleted antioxidants. The treatment with AS-AgNPs considerably elevated the caspase-9 and -3 activities and Bax expression, while decreasing the Bcl-2 expression in MCF-7 cells. Hence the current investigation reports that the formulated AS-AgNPs exhibited remarkable in vitro anticancer action against MCF-7 cells through increased ROS, oxidative stress, and apoptotic protein expression. The fabricated AS-AgNPs could be a possible anticancer remedy in the future.

Characterization of pectin-xylan-glucan-arabinogalactan proteins complex from Siberian fir Abies sibirica Ledeb.

Polysaccharide ASK was isolated from the Abies sibirica foliage by extraction with an aqueous KOH solution. ASK was shown to contain structurally different polymers such as arabinoglucuronoxylans, xyloglucans, glucomannans, arabinogalactan-proteins (AGPs). The pectic polysaccharides were also found in the alkaline extract of ASK and were represented by regions of homogalactorunan and rhamnogalactouronan-I whose side sugar chains were made up chiefly of highly branched 1,5-α-l-arabinan. The potential couplings between those polysaccharides were examined. Our studies showed simultaneous elution of pectin, xyloglucans, arabinoglucuronoxylans and AGPs, indicating that pectins can be covalently bound to the other cell-wall polysaccharides. NMR spectroscopy results revealed that the polysaccharides obtained by ion-exchange chromatography almost had no free reducing ends. These findings corroborate the conclusion that pectin, AGPs, glucan and xylan are bound together. The existence of the covalently bound complex of pectin-xylan-xyloglucan-AGP is suggested herein. Pectin and xylan are hypothesized to be covalently linked through RG-I regions.

Weather and climate controls over the seasonal carbon isotope dynamics of sugars from subalpine forest trees.

We examined the environmental variables that influence the delta(13)C value of needle and phloem sugars in trees in a subalpine forest. We collected sugars from Pinus contorta, Picea engelmannii and Abies lasiocarpa from 2006 to 2008. Phloem and needle sugars were enriched in (13)C during the autumn, winter and early spring, but depleted during the growing season. We hypothesized that the late-winter and early-spring (13)C enrichment was due to the mobilization of carbon assimilated the previous autumn; however, needle starch concentrations were completely exhausted by autumn, and we observed evidence of new starch production during episodic warm weather events during the winter and early-spring. Instead, we found that (13)C enrichment was best explained by the occurrence of cold night-time temperatures. We also observed seasonal decoupling in the (13)C/(12)C ratios of needle and phloem sugars. We hypothesized that this was due to seasonally-changing source-sink patterns, which drove carbon translocation from the needles towards the roots early in the season, before bud break, but from the roots towards the needles later in the season, after bud break. Overall, our results demonstrate that the (13)C/(12)C ratio of recently-assimilated sugars can provide a sensitive record of the short-term coupling between climate and tree physiology.

Tree species effects on ecosystem water-use efficiency in a high-elevation, subalpine forest.

Ecosystem water-use efficiency (eWUE; the ratio of net ecosystem productivity to evapotranspiration rate) is a complex landscape-scale parameter controlled by both physical and biological processes occurring in soil and plants. Leaf WUE (lWUE; the ratio of leaf CO(2) assimilation rate to transpiration rate) is controlled at short time scales principally by leaf stomatal dynamics and this control varies among plant species. Little is known about how leaf-scale variation in lWUE influences landscape-scale variation in eWUE. We analyzed approximately seven thousand 30-min averaged eddy covariance observations distributed across 9 years in order to assess eWUE in two neighboring forest communities. Mean eWUE was 19% lower for the community in which Engelmann spruce and subalpine fir were dominant, compared to the community in which lodgepole pine was dominant. Of that 19% difference, 8% was attributed to residual bias in the analysis that favored periods with slightly drier winds for the spruce-fir community. In an effort to explain the remaining 11% difference, we assessed patterns in lWUE using C isotope ratios. When we focused on bulk tissue from older needles we detected significant differences in lWUE among tree species and between upper and lower canopy needles. However, when these differences were scaled to reflect vertical and horizontal leaf area distributions within the two communities, they provided no power to explain differences in eWUE that we observed in the eddy covariance data. When we focused only on bulk needle tissue of current-year needles for 3 of the 9 years, we also observed differences in lWUE among species and in needles from upper and lower parts of the canopy. When these differences in lWUE were scaled to reflect leaf area distributions within the two communities, we were able to explain 6.3% of the differences in eWUE in 1 year (2006), but there was no power to explain differences in the other 2 years (2003 and 2007). When we examined sugars extracted from needles at 3 different times during the growing season of 2007, we could explain 3.8-6.0% of the differences in eWUE between the two communities, but the difference in eWUE obtained from the eddy covariance record, and averaged over the growing season for this single year, was 32%. Thus, overall, after accounting for species effects on lWUE, we could explain little of the difference in eWUE between the two forest communities observed in the eddy covariance record. It is likely that water and C fluxes from soil, understory plants, and non-needle tissues, account for most of the differences observed in the eddy covariance data. For those cases where we could explain some of the difference in eWUE on the basis of species effects, we partitioned the scaled patterns in lWUE into two components: a component that is independent of canopy leaf area distribution, and therefore only dependent on species-specific differences in needle physiology; and a component that is independent of species differences in needle physiology, and only dependent on species-specific influences on canopy leaf area distribution. Only the component that is dependent on species influences on canopy leaf area distribution, and independent of inherent species differences in needle physiology, had potential to explain differences in eWUE between the two communities. Thus, when tree species effects are important, canopy structure, rather than species-specific needle physiology, has more potential to explain patterns in eWUE.

Incorporation and remobilization of ¹³C within the fine-root systems of individual Abies alba trees in a temperate coniferous stand.

Forest ecosystems have a large carbon (C) storage capacity, which depends on their productivity and the residence time of C. Therefore, the time interval between C assimilation and its return to the atmosphere is an important parameter for determining C storage. Especially fine roots (≤2 mm in diameter) undergo constant replacement and provide a large biomass input to the soil. In this study, we aimed to determine the residence time of C in living fine roots and the decomposition rates of dead fine roots. Therefore, we pulse-labelled nine 20-year-old individual silver fir trees (Abies alba Miller; ∼70 cm tall) with ¹³CO2 in situ to trace the assimilated C over time into the fine-root systems. Whole trees were harvested at different time points after labelling in autumn, biomass was determined and cellulose and starch of fine roots were extracted. Moreover, soil cores were taken and ingrowth cores installed, in which fine roots were genetically identified, to assess incorporation and remobilization of ¹³C in the fine roots of silver fir trees; litterbags were used to determine fine-root decomposition rates. The ¹³C label was incorporated in the fine-root system as cellulose within 3 days, with highest values after 30 days, before reaching background levels after 1 year. The highest δ¹³C values were found in starch throughout the experiment. ¹³C recovery and carbon mean residence times did not differ significantly among fine-root diameter classes, indicating size-independent C turnover times in fine roots of A. alba trees of ∼219 days. Furthermore, carbon was remobilized from starch into newly grown fine roots in the next spring after our autumn labelling. One year after installation, litterbags with fine roots revealed a decrease of biomass of ∼40% with relative ¹³C content in fine-root bulk biomass and cellulose of ∼50%, indicating a faster loss of ¹³C-labelled compounds compared with bulk biomass. Our results also suggest that genetic analysis of fine-root fragments found in soil and ingrowth cores is advisable when working in mixed forest stands with trees of similar fine-root morphology. Only then can one avoid dilution of the labelling signal by mistake, due to analysis of non-labelled non-target species roots.

Autotrophic and heterotrophic respiration in needle fir and Quercus-dominated stands in a cool-temperate forest, central Korea.

To investigate annual variation in soil respiration (R (S)) and its components [autotrophic (R (A)) and heterotrophic (R (H))] in relation to seasonal changes in soil temperature (ST) and soil water content (SWC) in an Abies holophylla stand (stand A) and a Quercus-dominated stand (stand Q), we set up trenched plots and measured R (S), ST and SWC for 2 years. The mean annual rate of R (S) was 436 mg CO(2) m(-2) h(-1), ranging from 76 to 1,170 mg CO(2) m(-2) h(-1), in stand A and 376 mg CO(2) m(-2) h(-1), ranging from 82 to 1,133 mg CO(2) m(-2) h(-1), in stand Q. A significant relationship between R (S) and its components and ST was observed over the 2 years in both stands, whereas a significant correlation between R (A) and SWC was detected only in stand Q. On average over the 2 years, R (A) accounted for approximately 34% (range 17-67%) and 31% (15-82%) of the variation in R (S) in stands A and Q, respectively. Our results suggested that vegetation type did not significantly affect the annual mean contributions of R (A) or R (H), but did affect the pattern of seasonal change in the contribution of R (A) to R (S).

Sugar, amino acid and inorganic ion profiling of the honeydew from different hemipteran species feeding on Abies alba and Picea abies.

Several hemipteran species feed on the phloem sap of plants and produce large amounts of honeydew that is collected by bees to produce honeydew honey. Therefore, it is important to know whether it is predominantly the hemipteran species or the host plant to influence the honeydew composition. This is particularly relevant for those botanical and zoological species from which the majority of honeydew honey originates. To investigate this issue, honeydew from two Cinara species located on Abies alba as well as from two Cinara and two Physokermes species located on Picea abies were collected. Phloem exudates of the host plants were also analyzed. Honeydew of all species contained different proportions of hexoses, sucrose, melezitose, erlose, and further di- and trisaccharides, whereas the phloem exudates of the host trees contained no trisaccharides. Moreover, the proportions of sugars differed significantly between hemipteran species feeding on the same tree species. Sucrose hydrolysis and oligosaccharide formation was shown in whole-body homogenates of aphids. The type of the produced oligosaccharides in the aphid-extracts correlated with the oligosaccharide composition in the honeydew of the different aphid species. The total contents of amino acids and inorganic ions in the honeydew were much lower than the sugar content. Glutamine and glutamate were predominant amino acids in the honeydew of all six hemipteran species and also in the phloem exudates of both tree species. Potassium was the dominant inorganic ion in all honeydew samples and also in the phloem exudate. Statistical analyses reveal that the sugar composition of honeydew is determined more by the hemipteran species than by the host plant. Consequently, it can be assumed that the sugar composition of honeydew honey is also more influenced by the hemipteran species than by the host tree.

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.

Competition and drought limit the response of water-use efficiency to rising atmospheric carbon dioxide in the Mediterranean fir Abies pinsapo.

The gas-exchange and radial growth responses of conifer forests to climatic warming and increasing atmospheric CO2 have been widely studied. However, the modulating effects of variables related to stand structure (e.g., tree-to-tree competition) on those responses are poorly explored. The basal-area increment (BAI) and C isotope discrimination (C stable isotope ratio; delta13C) in the Mediterranean fir Abies pinsapo were investigated to elucidate the influences of stand competition, atmospheric CO2 concentrations and climate on intrinsic water-use efficiency (WUEi). We assessed the variation in delta13C of tree-rings from dominant or co-dominant trees subjected to different degrees of competition. A high- (H) and a low-elevation (L) population with contrasting climatic constraints were studied in southern Spain. Both populations showed an increase in long-term WUEi. However, this increase occurred more slowly at the L site, where a decline of BAI was also observed. Local warming and severe droughts have occurred in the study area over the past 30 years, which have reduced water availability more at lower elevations. Contrastingly, trees from the H site were able to maintain high BAI values at a lower cost in terms of water consumption. In each population, trees subjected to a higher degree of competition by neighboring trees showed lower BAI and WUEi than those subjected to less competition, although the slopes of the temporal trends in WUEi were independent of the competitive micro-environment experienced by the trees. The results are consistent with an increasing drought-induced limitation of BAI and a decreasing rate of WUEi improvement in low-elevation A. pinsapo forests. This relict species might not be able to mitigate the negative effects of a decrease in water availability through a reduction in stomatal conductance, thus leading to a growth decline in the more xeric sites. An intense and poorly asymmetric competitive environment at the stand level may also act as an important constraint on the adaptive capacity of these drought-sensitive forests to climatic warming.

Seasonal changes in abundance and phosphorylation status of photosynthetic proteins in eastern white pine and balsam fir.

During winter, the light-harvesting complexes of evergreen plants change function from energy-harvesting to energy-dissipating centers. The goal of our study was to monitor changes in the composition of the photosynthetic apparatus that accompany these functional changes. Seasonal changes in chlorophyll fluorescence, pigment concentration, and abundance and phosphorylation status of photosynthetic proteins in Pinus strobus L. (sun-exposed trees) and Abies balsamea (L.) P. Mill. (sun-exposed and shaded trees) were examined in the cold winter climate of Minnesota. Results indicated typical seasonal changes in chlorophyll fluorescence and pigment concentration, with sustained reduced photosystem II (PSII) efficiency during winter, accompanied by retention of zeaxanthin and antheraxanthin, and winter increases in the pool of xanthophyll cycle pigments and lutein. In sun-exposed trees, all photosynthetic proteins that were monitored decreased in relative abundance during winter, although two light-harvesting chlorophyll a/b binding proteins (Lhcb2 and Lhcb5), and the PsbS protein, were enriched in non-summer months, suggesting a role for these proteins in winter acclimation. In contrast, shaded trees maintained most of their protein throughout winter, with reductions occurring in spring. Thylakoid protein phosphorylation data suggest winter increases in the phosphorylation of a PSII core protein, PsbH, in sun-exposed trees, and increases in phosphorylation of all PSII core proteins in shaded trees.

Temporal variation of nonstructural carbohydrates in montane conifers: similarities and differences among developmental stages, species and environmental conditions.

Nonstructural carbohydrates (NSCs) are commonly used to assess the balance of carbon sources and sinks in plants. A notable application of this approach has been tests of hypotheses on carbon limitations of trees at their upper altitudinal limits, near the alpine. How NSCs vary in time is not well known in conifers during their critical seedling stage, despite the importance of knowing the temporal variations of NSCs to use snapshot measurements of NSCs to assess carbon balance. We measured NSCs in needles, separately as soluble sugars and starch; (1) over diurnal periods in seedlings of Pseudotsuga menziesii (Mirb.) Franco (a timberline species that does not occur up to treeline), (2) throughout the growth season in the seedlings of P. menziesii and Abies lasiocarpa (Hook.) Nutt. (a species that does occur up to treeline) growing along an elevation gradient in the timberline ecotone and furthermore (3) compared seedlings and co-occurring adults to assess variation with developmental stage. We also compared NSCs in seedlings grown under field or laboratory conditions to separate environmental from intrinsic factors affecting NSCs during early emergence. Diurnal variations in NSCs were minimal, especially when compared to seasonal variation, and were detectable mainly in relatively small midday maxima of soluble sugar concentrations. Seasonal patterns of NSCs were generally (and surprisingly) similar among field and laboratory seedlings and adults. Seasonal patterns of NSCs were dominated by progressive increases in soluble sugars until winter, and by early-season peaks in starch. Nonetheless, notable differences were detectable among ages, species and environmental conditions in (1) the timing and extent of the early-season maxima of starch and (2) the extent of the late-season maxima of soluble sugars. These differences in NSCs likely correspond with ecophysiologically relevant differences in carbon balance that could affect growth and survival of trees growing in the timberline ecotone.