ALBERT

Description: Much evidence show that over-expression of epidermal growth factor receptor (EGFR) plays an important role in regulating carcinogenesis. Genetic variations in 3' untranslated region (3'UTR) of gene have been reported to affect gene expression by interfering with microRNAs (miRNAs), which are thought to function as either tumour suppressors or oncogenes by binding to their target mRNA. In this study, we investigated the association between the EGFR 3'UTR 774T〉C polymorphism and bladder cancer risk. We used the TaqMan technology to genotype this genetic variant in a hospital-based case–control study of 908 bladder cancer patients and 1239 controls in a Chinese population. We found that the 774CC genotype was associated with a statistically significantly increased risk of bladder cancer [adjusted odds ratio = 1.29, 95% confidence interval = 1.05–1.58], compared with the 774TT/TC genotype, and this increased risk was more pronounced among subgroups of age 〉 65 years, non-smokers and patients’ tumour invasive stage. Furthermore, luciferase assays in T24 cell showed that EGFR 3'UTR 774 T to C substitution could increase the expression of EGFR, which was consistent with the association study finding. Additionally, we also provide evidence that 774T〉C polymorphism increasing EGFR expression was not regulated by hsa-miR-214 binding. These findings suggested that EGFR 3'UTR 774T〉C polymorphism may contribute to susceptibility to bladder cancer.

Description: The impact of stored water on estimates of transpiration from scaled sap flux measurements was assessed in mature Pinus taeda (L.) at the Duke Free-Air CO 2 Enrichment (FACE) site. We used a simple hydraulic model with measurements of sap flux ( J ) at breast height and the base of the live crown for 26 trees over 6 months to examine the effects of elevated CO 2 (eCO 2 ) and fertilization (N F ) treatments, as well as temporal variation in soil moisture ( M ( t ) ), on estimates of the hydraulic time constant (). At low M ( t ) , there was little (〈12%) difference in of different treatments. At high M ( t ) , differences were much greater, with reductions of 27, 52 and 34% in eCO 2 , N F and eCO 2 x N F respective to the control. Incorporating with these effects into the analysis of a larger data set of previous J measurements at this site (1998–2008) improved agreement between modeled and measured values in 92% of cases. However, a simplified calibration of that neglected treatment and soil moisture effects performed more dependably, improving agreement in 98% of cases. Incorporating had the effect of increasing estimates of reference stomatal conductance at 1 kPa vapor pressure deficit (VPD) and saturating photosynthetic active radiation (PAR) an average of 12–14%, while increasing estimated sensitivities to VPD and PAR. A computationally efficient hydraulic model, such as the one presented here, incorporated into a hierarchical model of stomatal conductance presents a novel approach to including hydraulic time constants in estimates of stomatal responses from long-term sap flux data sets.

Description: Inferences on climate change effects are reliable only if they are based on a causal relationship rather than simple statistical predictive capacity. To assess for causal links between climate and mature black spruce ( Picea mariana (Mills.) BSP) radial growth, we combined the use of wood anatomy, cambium phenology, climate and soil measurements (air temperature and humidity, precipitations, soil temperature and water content, photosynthetically active radiation), and a model selection approach proceeding backwards from a full model. Results show that the number of tracheids is responsible for 88% of the variation in ring width whereas mean tracheid diameter accounts for the remaining 12%. The number of tracheids produced depends on factors related to photosynthesis during tracheid production, i.e., daily light intensity and maximum temperature between the day of initiation and the day of cessation of tracheid production, plus soil temperature during August of the previous year which is an important period for determining the number of new needles produced. It is also important to consider duration of the period for tracheid production. These results imply that short-term climate change should increase black spruce radial growth. They also suggest that the typical use of post-growth ring width sampling individually linked to air temperature and precipitations is not sufficient to infer climate change effects accurately on radial growth where there is no strong single climatic limitation but multiple limitations instead.

Description: It is generally assumed that the largest vessels are occurring in the roots and that vessel diameters and the related hydraulic conductance in the xylem are decreasing acropetally from roots to leaves. With this study in five tree species of a perhumid tropical rainforest in Sulawesi (Indonesia), we searched for patterns in hydraulic architecture and axial conductivity along the flow path from small-diameter roots through strong roots and the trunk to distal sun-canopy twigs. Wood density differed by not more than 10% across the different flow path positions in a species, and branch and stem wood density were closely related in three of the five species. Other than wood density, the wood anatomical and xylem hydraulic traits varied in dependence on the position along the flow path, but were unrelated to wood density within a tree. In contrast to reports from conifers and certain dicotyledonous species, we found a hump-shaped variation in vessel diameter and sapwood area-­specific conductivity along the flow path in all five species with a maximum in the trunk and strong roots and minima in both small roots and twigs; the vessel size depended on the diameter of the organ. This pattern might be an adaptation to the perhumid climate with a low risk of hydraulic failure. Despite a similar mean vessel diameter in small roots and twigs, the two distal organs, hydraulically weighted mean vessel diameters were on average 30% larger in small roots, resulting in ~ 85% higher empirical and theoretical specific conductivities. Relative vessel lumen area in percent of sapwood area decreased linearly by 70% from roots to twigs, reflecting the increase in sclerenchymatic tissue and tracheids in acropetal direction in the xylem. Vessel size was more closely related to the organ diameter than to the distance along the root-to-shoot flow path. We conclude that (i) the five co-occurring tree species show convergent patterns in their hydraulic architecture despite different growth strategies, and (ii) the paradigm assuming continuous acropetal vessel tapering and decrease in specific conductance from fine roots towards distal twigs needs reconsideration.

Description: In this study, we employ a network of thermal dissipation probes (TDPs) monitoring sap flux density to estimate leaf-specific transpiration ( E L ) and stomatal conductance ( G S ) in Pinus taeda (L.) and Liquidambar styraciflua L. exposed to +200 ppm atmospheric CO 2 levels (eCO 2 ) and nitrogen fertilization. Scaling half-hourly measurements from hundreds of sensors over 11 years, we found that P. taeda in eCO 2 intermittently (49% of monthly values) decreased stomatal conductance ( G S ) relative to the control, with a mean reduction of 13% in both total E L and mean daytime G S . This intermittent response was related to changes in a hydraulic allometry index ( A H ), defined as sapwood area per unit leaf area per unit canopy height, which decreased a mean of 15% with eCO 2 over the course of the study, due mostly to a mean 19% increase in leaf area ( A L ). In contrast, L. styraciflua showed a consistent (76% of monthly values) reduction in G S with eCO 2 with a total reduction of 32% E L , 31% G S and 23% A H (due to increased A L per sapwood area). For L. styraciflua , like P. taeda , the relationship between A H and G S at reference conditions suggested a decrease in G S across the range of A H . Our findings suggest an indirect structural effect of eCO 2 on G S in P. taeda and a direct leaf level effect in L. styraciflua . In the initial year of fertilization, P. taeda in both CO 2 treatments, as well as L. styraciflua in eCO 2 , exhibited higher G S with N F than expected from shifts in A H , suggesting a transient direct effect on G S . Whether treatment effects on mean leaf-specific G S are direct or indirect, this paper highlights that long-term treatment effects on G S are generally reflected in A H as well.

Description: Estimating sapwood area is one of the main sources of error when upscaling point scale sap flow measurements to whole-tree water use. In this study, the potential use of electrical resistivity tomography (ERT) to determine the sapwood–heartwood (SW–HW) boundary is investigated for Pinus elliottii Engelm var. elliottii x Pinus caribaea Morelet var. hondurensis growing in a subtropical climate. Specifically, this study investigates: (i) how electrical resistivity is correlated to either wood moisture content, or electrolyte concentration, or both, and (ii) how the SW–HW boundary is defined in terms of electrical resistivity. Tree cross-sections at breast height are analysed using ERT before being felled and the cross-section surface sampled for analysis of major electrolyte concentrations, wood moisture content and density. Electrical resistivity tomography results show patterns with high resistivities occurring in the inner part of the cross-section, with much lower values towards the outside. The high-resistivity areas were generally smaller than the low-resistivity areas. A comparison between ERT and actual SW area measured after felling shows a slope of the linear regression close to unity (=0.96) with a large spread of values ( R 2 = 0.56) mostly due to uncertainties in ERT. Electrolyte concentrations along sampled radial transects (cardinal directions) generally showed no trend from the centre of the tree to the bark. Wood moisture content and density show comparable trends that could explain the resistivity patterns. While this study indicates the potential for application of ERT for estimating SW area, it shows that there remains a need for refinement in locating the SW–HW boundary (e.g., by improvement of the inversion method, or perhaps electrode density) in order to increase the robustness of the method.

Description: Palms are an important component of tropical ecosystems, living alongside dicotyledonous trees, even though they have a very different growth pattern and vascular system. As monocots, vessels in palms are located within vascular bundles and, without a vascular cambium that many dicotyledonous trees possess, palms cannot add additional vessels to their vascular system as they get older and taller. This means that hydraulic architecture in palms is more predetermined, which may require a highly efficient hydraulic system. This preset nature, along with the decoupling of hydraulic and mechanical functioning to different cell types, may allow palms to have a more efficient hydraulic system than dicotyledonous trees. Therefore, this study seeks to determine the efficiency of the hydraulic system in the palm Iriartea deltoidea (Ruiz & Pav.) and compare this efficiency with other tree forms. We measured cross-sectional areas of roots, stems and fronds as well as leaf areas of I. deltoidea saplings. Likewise, cross-sections were made and vessel diameters and frequencies measured. This allowed for the calculation of theoretical specific conductivity ( K S,calc ), theoretical leaf-specific conductivity ( K L,calc ), and vessel diameter and vessel number ratios between distal and proximal locations in the palms. Iriartea deltoidea palms were found to have the largest, least frequent vessels that diverged most from the square packing limit (maximum number of vessels that fit into a given area) compared with other major tree forms, and they therefore invested the least space and carbon into water transport structures. Likewise, conduits tapered by ~1/3 between ranks (root, bole and petiole), which represents an efficient ratio with regard to the trade-offs between safety and efficiency of the conducting system. Conduits also exhibited a high conservation of the sum of the conduit radii cubed ( r 3 ) across ranks, thereby approximating Murray's law patterning. Therefore, our results indicate that the palm I. deltoidea has a very efficient hydraulic system in terms of maintaining a large conducting capacity with a minimal vascular investment. This efficiency may allow palms to compete well with dicotyledonous trees in tropical and subtropical climates but other developmental factors largely restrict palms from regions that experience prolonged freezing temperatures.

Description: Functional attributes determine the survival and growth of planted seedlings in reforestation projects. Nitrogen (N) and water are important resources in the cultivation of forest species, which have a strong effect on plant functional traits. We analyzed the influence of N nutrition on drought acclimation of Pinus pinea L. seedlings. Specifically, we addressed if high N fertilization reduces drought and frost tolerance of seedlings and whether drought hardening reverses the effect of high N fertilization on stress tolerance. Seedlings were grown under two N fertilization regimes (6 and 100 mg N per plant) and subjected to three drought-hardening levels (well-watered, moderate and strong hardening). Water relations, gas exchange, frost damage, N concentration and growth at the end of the drought-hardening period, and survival and growth of seedlings under controlled xeric and mesic outplanting conditions were measured. Relative to low-N plants, high-N plants were larger, had higher stomatal conductance (27%), residual transpiration (11%) and new root growth capacity and closed stomata at higher water potential. However, high N fertilization also increased frost damage (24%) and decreased plasmalemma stability to dehydration (9%). Drought hardening reversed to a great extent the reduction in stress tolerance caused by high N fertilization as it decreased frost damage, stomatal conductance and residual transpiration by 21, 31 and 24%, respectively, and increased plasmalemma stability to dehydration (8%). Drought hardening increased tissue non-structural carbohydrates and N concentration, especially in high-fertilized plants. Frost damage was positively related to the stability of plasmalemma to dehydration ( r = 0.92) and both traits were negatively related to the concentration of reducing soluble sugars. No differences existed between moderate and strong drought-hardening treatments. Neither N nutrition nor drought hardening had any clear effect on seedling performance under xeric outplanting conditions. However, fertilization increased growth under mesic conditions, whereas drought hardening decreased growth. We conclude that drought hardening and N fertilization applied under typical container nursery operational conditions exert opposite effects on the physiological stress tolerance of P. pinea seedlings. While drought hardening increases overall stress tolerance, N nutrition reduces it and yet has no effect on the drought acclimation capacity of seedlings.

Description: We investigated Scots pine adaptive responses to the light spectra by measuring hypocotyl length in seeds sampled from three natural Scots pine ecotypes across a latitudinal cline ranging from 63° to 68° N in Sweden where the adaptive cline is known to be steeper. Seeds were germinated under dark (D) and three monochromatic continuous light wavelengths: blue (B), red (R) and far-red (FR). Analysis of variance revealed a northward decrease in the inhibitory effect of FR with respect to D, the so-called far red high irradiance response. Ecotypic variation for hypocotyl development was observed under the FR and D treatments, while the trends for the B and R treatments were not statistically significant. Under FR the ecotypic variation showed an increase in hypocotyl length northwards, in contrast to the treatment under D which showed a decrease in the hypocotyl length northwards. These results could be interpreted in view of the previously reported northward increase in FR requirement to maintain growth in Norway spruce and Scots pine. Prior to the performance of the main light experiment, the maternal effect on progeny performance was investigated, which showed the absence of maternal environment effect on the performance of the seedlings.

Description: Drought stress can induce closure of stomata, thus leading to photoinhibition. The effects of prolonged severe drought under natural growing conditions on photosystem I (PSI), photosystem II (PSII) and cyclic electron flow (CEF) in drought-tolerant tree species are unclear. In spring 2010, southwestern China confronted severe drought that lasted several months. Using three dominant evergreen species, Cleistanthus sumatranus (Miq.) Muell. Arg. (Euphorbiaceae), Celtis philippensis Bl. (Ulmaceae) and Pistacia weinmannifolia J. Poisson ex Franch. (Anacardiaceae) that are native to a tropical limestone forest, we investigated the influence of this stress on PSI and PSII activities as well as light energy distribution in the PSII and P700 redox state. By the end of the drought period, predawn leaf water potential ( pd ) largely declined in each species, especially in C. sumatranus . Photosystem I activity strongly decreased in the three species, especially in C. sumatranus which showed a decrease of 65%. The maximum quantum yield of PSII after dark adaptation remained stable in P. weinmannifolia and C. philippensis but significantly decreased in C. sumatranus . Light response curves indicated that both linear electron flow and non-photochemical quenching were severely inhibited in C. sumatranus along with disappearance of CEF, resulting in deleterious excess light energy in PSII. We conclude that PSI is more sensitive than PSII to prolonged severe drought in these three drought-tolerant species, and CEF is essential for photoprotection in them.

Description: We investigated changes in chlorophyll a fluorescence from alternate leaf surfaces to assess the intraleaf light acclimation patterns in combination with natural variations in radiation, leaf angles, leaf mass per area (LMA), chlorophyll content (Chl) and leaf optical parameters. Measurements were conducted on bottom- and top-layer leaves of Tilia cordata Mill. (a shade-tolerant sub-canopy species, sampled at heights of 11 and 16 m) and Populus tremula L. (a light-demanding upper canopy species, sampled at canopy heights of 19 and 26 m). The upper canopy species P. tremula had a six times higher PSII quantum yield ( II ) and ratio of open reaction centres (qP), and a two times higher LMA than T. cordata . These species-specific differences were also present when the leaves of both species were in similar light conditions. Leaf adaxial/abaxial fluorescence ratio was significantly larger in the case of more horizontal leaves. Populus tremula (more vertical leaves), had smaller differences in fluorescence parameters between alternate leaf sides compared with T. cordata (more horizontal leaves). However, optical properties on alternate leaf sides showed a larger difference for P. tremula . Intraspecifically, the measured optical parameters were better correlated with LMA than with leaf Chl. Species-specific differences in leaf anatomy appear to enhance the photosynthetic potential of leaf biochemistry by decreasing the interception of excess light in P. tremula and increasing the light absorptance in T. cordata . Our results indicate that intraleaf light absorption gradient, described here as leaf adaxial/abaxial side ratio of chlorophyll a fluorescence, varies significantly with changes in leaf light environment in a multi-layer multi-species tree canopy. However, this variation cannot be described merely as a simple function of radiation, leaf angle, Chl or LMA, and species-specific differences in light acclimation strategies should also be considered.

Description: Norway maple ( Acer platanoides L), which is among the most invasive tree species in forests of eastern North America, is associated with reduced regeneration of the related native species, sugar maple ( Acer saccharum Marsh) and other native flora. To identify traits conferring an advantage to Norway maple, we grew both species through an entire growing season under simulated light regimes mimicking a closed forest understorey vs. a canopy disturbance (gap). Dynamic shade-houses providing a succession of high-intensity direct-light events between longer periods of low, diffuse light were used to simulate the light regimes. We assessed seedling height growth three times in the season, as well as stem diameter, maximum photosynthetic capacity, biomass allocation above- and below-ground, seasonal phenology and phenotypic plasticity. Given the north European provenance of Norway maple, we also investigated the possibility that its growth in North America might be increased by delayed fall senescence. We found that Norway maple had significantly greater photosynthetic capacity in both light regimes and grew larger in stem diameter than sugar maple. The differences in below- and above-ground biomass, stem diameter, height and maximum photosynthesis were especially important in the simulated gap where Norway maple continued extension growth during the late fall. In the gap regime sugar maple had a significantly higher root : shoot ratio that could confer an advantage in the deepest shade of closed understorey and under water stress or browsing pressure. Norway maple is especially invasive following canopy disturbance where the opposite (low root : shoot ratio) could confer a competitive advantage. Considering the effects of global change in extending the potential growing season, we anticipate that the invasiveness of Norway maple will increase in the future.

Description: Fungal colonization can significantly affect the secondary metabolism of the host plants. We tested the impact of a common below-ground symbiosis, i.e., ectomycorrhiza formation, on poplar leaf chemical components that are involved in the defence against a common disease, i.e., rust fungi, in N-deficient soil. A rust-susceptible poplar clone ( Populus trichocarpa x deltoides ‘Beaupré’) was (a) non-associated with ectomycorrhizal fungus (EM) Hebeloma mesophaeum (Pers.) Quélet MÜN and non-infected with rust fungus Melampsora larici-populina Kleb. (isolate 98AG31), (b) associated with EM, (c) inoculated with rust fungus and (d) associated with EM and inoculated with rust fungus. Poplar leaves were analysed by photometric and mass spectrometric techniques (liquid chromatography–tandem mass spectrometry (LC-MS/MS), pyrolysis–field ionization mass spectrometry (Py-FIMS)). Both rust infection and mycorrhiza formation led to increased proportions of condensed tannins in relation to total phenolics (13% in the control, 18–19% in the fungal treatments). In contrast, salicylic acid concentration (6.8 µg g –1 in the control) was higher only in the rust treatments (17.9 and 25.4 µg g –1 with rust infection). The Py-FIMS analysis revealed that the rust-infected treatments were significantly separated from the non-rust-infected treatments on the basis of six flavonoids and one lipid. The relative abundance of these components, which have known functions in plant defence, was decreased after rust infection of non-mycorrhizal plants, but not in mycorrhizal plants. The results indicate that the ectomycorrhizal formation compensated the rust infection by a decrease in the flavonoid syntheses. The study provides new evidence for an interactive response of mycorrhizal colonization and infection with rust fungi in the metabolism of poplar.

Description: Apple proliferation (AP) represents a serious threat to several fruit-growing areas and is responsible for great economic losses. Several studies have highlighted the key role played by the cell wall in response to pathogen attack. The existence of a cell wall integrity signaling pathway which senses perturbations in the cell wall architecture upon abiotic/biotic stresses and activates specific defence responses has been widely demonstrated in plants. More recently a role played by cell wall-related genes has also been reported in plants infected by phytoplasmas. With the aim of shedding light on the cell wall response to AP disease in the economically relevant fruit-tree Malus x domestica Borkh., we investigated the expression of the cellulose ( CesA ) and callose synthase ( CalS ) genes in different organs (i.e., leaves, roots and branch phloem) of healthy and infected symptomatic outdoor-grown trees, sampled over the course of two time points (i.e., spring and autumn 2011), as well as in in vitro micropropagated control and infected plantlets. A strong up-regulation in the expression of cell wall biosynthetic genes was recorded in roots from infected trees. Secondary cell wall CesA s showed up-regulation in the phloem tissue from branches of infected plants, while either a down-regulation of some genes or no major changes were observed in the leaves. Micropropagated plantlets also showed an increase in cell wall-related genes and constitute a useful system for a general assessment of gene expression analysis upon phytoplasma infection. Finally, we also report the presence of several ‘knot’-like structures along the roots of infected apple trees and discuss the occurrence of this interesting phenotype in relation to the gene expression results and the modalities of phytoplasma diffusion.

Description: The availability of a system for direct transfer of antifungal candidate genes into European chestnut ( Castanea sativa Mill.) would offer an alternative approach to conventional breeding for production of chestnut trees tolerant to ink disease caused by Phytophthora spp. For the first time, a chestnut thaumatin-like protein gene ( CsTL1 ), isolated from chestnut cotyledons, has been overexpressed in three chestnut somatic embryogenic lines. Transformation experiments have been performed using an Agrobacterium tumefaciens Smith and Townsend vector harboring the neomycin phosphotransferase ( NPTII ) selectable and the green fluorescent protein ( EGFP ) reporter genes. The transformation efficiency, determined on the basis of the fluorescence of surviving explants, was clearly genotype dependent and ranged from 32.5% in the CI-9 line to 7.1% in the CI-3 line. A total of 126 independent transformed lines were obtained. The presence and integration of chestnut CsTL1 in genomic DNA was confirmed by polymerase chain reaction (PCR) and Southern blot analyses. Quantitative real-time PCR revealed that CsTL1 expression was up to 13.5-fold higher in a transgenic line compared with its corresponding untransformed line. In only one of the 11 transformed lines tested, expression of the CsTL1 was lower than the control. The remaining 115 transformed lines were successfully subjected to cryopreservation. Embryo proliferation was achieved in all of the transgenic lines regenerated and the transformed lines showed a higher mean number of cotyledonary stage embryos and total number of embryos per embryo clump than their corresponding untransformed lines. Transgenic plants were regenerated after maturation and germination of transformed somatic embryos. Furthermore, due to the low plantlet conversion achieved, axillary shoot proliferation cultures were established from partially germinated embryos (only shoot development), which were multiplied and rooted according to procedures already established. Transgenic plants were acclimatized and grown in a greenhouse. No phenotypic differences were found with control plants, suggesting no potential cytotoxic effects of the green fluorescent protein. The results reported in the present work could be considered as a first step toward the production of fungal-disease tolerant cisgenic chestnut plants.

Description: Studying the dynamics of stem water content (   ) in living trees has an outstanding physiological interest but all the available techniques to measure exhibit major drawbacks. In this work, we present a new methodology to estimate variations in along with sap velocity using the compensated heat pulse (CHP) technique. One lab experiment was performed on several wooden blocks obtained from three different tree species. Samples were slowly dried and their moisture loss was monitored by both gravimetric approaches and time-domain reflectometry (TDR) or CHP probes in order to contrast the validity of our methodology (volumetric specific heat (VSH)-CHP) over a range of water contents. In addition, a field experiment was conducted to monitor fluctuations in standing olive trees ( Olea europaea L. cv. ‘Arbequina’) growing under three different irrigation regimes. In the lab test, the actual values deduced gravimetrically differed from the estimates yielded by the VSH-CHP method. However, it could successfully track relative changes in the water stored for the range of expected in living wood. Furthermore, the field experiment showed a seasonal change in , which was similar in shape and magnitude to those reported in the literature for olive and other Mediterranean tree species. On the other hand, differences in the seasonal patterns of between irrigation treatments strongly corresponded with those of sap flow and some leaf water potential measurements. The results of this work suggest that the CHP technique could be employed to monitor the dynamics of both and sap flow simultaneously in standing trees and evidence that seasonal changes in might be used as a long-term water status indicator.

Description: The emerald ash borer ( Agrilus planipennis Fairmaire, EAB) is an alien, invasive wood-boring insect that is responsible for killing millions of ash trees since its discovery in North America in 2002. All North American ash species ( Fraxinus spp.) that EAB has encountered have shown various degrees of susceptibility, while Manchurian ash ( Fraxinus mandshurica Ruprecht), which shares a co-evolutionary history with this insect, is resistant. Recent studies have looked into constitutive resistance mechanisms in Manchurian ash, concentrating on the secondary phloem, which is the feeding substrate for the insect. In addition to specialized metabolism and defense-related components, primary metabolites and nutritional summaries can also be important to understand the feeding behavior of insect herbivores. Here, we have compared the nutritional characteristics (water content, total protein, free amino acids, total soluble sugars and starch, percent carbon and nitrogen, and macro- and micronutrients) of outer bark and phloem from black, green, white and Manchurian ash to determine their relevance to resistance or susceptibility to EAB. Water content and concentrations of Al, Ba, Cu, Fe, K, Li, tryptophan and an unknown compound were found to separate black and Manchurian ash from green and white ash in a principal component analysis (PCA), confirming their phylogenetic placements into two distinct clades. The traits that distinguished Manchurian ash from black ash in the PCA were water content and concentrations of total soluble sugars, histidine, lysine, methionine, ornithine, proline, sarcosine, tyramine, tyrosol, Al, Fe, K, Na, V and an unknown compound. However, only proline, tyramine and tyrosol were significantly different, and higher, in Manchurian ash than in black ash.

Description: According to well-known biochemical and biophysical mechanisms, the stimulation of C 3 photosynthesis by elevated atmospheric CO 2 concentration ([CO 2 ]) is strongly modified by changes in temperature and radiation. In order to investigate whether a static parameterization of the commonly used Farquhar et al. model of photosynthesis (i.e., without CO 2 -induced seasonal or thermal acclimation of photosynthetic capacity) can accurately predict these interactions in mature boreal Norway spruce ( Picea abies (L.) Karst.) during the frost-free part of the growing season, shoot gas exchange was continuously measured on trees during their second/third year of exposure to ambient or doubled [CO 2 ] inside whole-tree chambers. The relative CO 2 -induced enhancement of net photosynthesis ( A n ) at a given temperature remained stable over the study period, but increased strongly with temperature and radiation, in agreement with predictions by the model. Light-saturated A n (+67% at 20 °C), dark respiration (+36%) and intercellular to ambient [CO 2 ] ratio ( c i / c a ; +27%) were significantly increased by CO 2 treatment. Stomatal conductance ( g s ) was not significantly affected. Our results demonstrate that the Farquhar et al. model of photosynthesis has the capability to predict interactions between [CO 2 ] and seasonal weather variability on A n in Norway spruce during the non-frost growing season without accounting for CO 2 -induced seasonal and/or thermal photosynthetic acclimation. However, stomatal model assumptions of reduced g s and constant c i / c a under rising atmospheric [CO 2 ] did not hold.

Description: Radial stem growth and the isotopic composition of growth rings are commonly used to quantify the effects of droughts on trees. However, often these parameters are quantified only at one stem height, e.g., 1.3 m, and it is not known how representative this is for the whole stem. This study investigated radial growth at four stem heights (1.3, 5.5, 9.8 and 14 m) of 21, and wood 13 C and 18 O at two heights (1.3 and 14 m) of 10 (co-)dominant Norway spruce trees from heavily (HT) and moderately thinned (MT) stands to assess whether different thinning intensities influenced the drought response of stems at different tree heights. Annual basal area increments (BAIs) and stable isotopes in earlywood and latewood were compared between thinning treatments and among the different stem heights. For BAIs, linear correlations with climate were analysed as well. The response of radial growth and isotopic composition to drought was similar at different stem heights in HT trees, but varied with height in MT trees, which were also more sensitive to climatic variations. Recovery of radial growth after drought was more rapid in trees from HT compared with MT stands, except for the topmost height. Basal area increments at breast height (1.3 m) provided good estimates of the volume growth response to drought for the whole stem, but not for its recovery. The faster recovery of radial growth at 1.3 m height of HT compared with MT trees after the 2003 drought was not accompanied by differences in recovery of isotopic composition. However, this is likely to be related to differences between treatments in remobilization of stored C and in tree structure (leaf area, root systems).

Description: Crown architecture is an important determinant of biomass production and yield of any bio-energy plantation since it determines leaf area display and hence light interception. Four Populus genotypes—of different species and hybrids and with contrasting productivity and leaf area—were examined in terms of their branch characteristics in relation to crown architecture during the first and second growing seasons after plantation establishment. The trees were planted at high density (8000 ha –1 ) on two different former land use types, cropland and pasture. We documented significant differences in branch architecture among the genotypes and for the first year among the former land use types. Land use effects only affected factors not related to canopy closure and wood production, and decreased after the first growing season. This suggested that both former land use types were equally suited for the establishment success of a poplar bio-energy plantation. Tree height and branch dimensions—branch diameter and branch length—were the most important determinants of wood production and maximum leaf area index. Despite the secondary importance of the number of sylleptic branches, these branches contributed significantly to the total leaf area in three out of the four studied genotypes. This indicated that enhanced syllepsis accelerates leaf area development and hence carbon assimilation, especially in the early stages of a high-density plantation with poplar.

Description: Polyamine (PA) metabolism was studied in liquid cultures of Scots pine ( Pinus sylvestris L.) embryogenic cells. The focus of the study was on the metabolic changes at the interphase between the initial lag phase and the exponential growth phase. PA concentrations fluctuated in the liquid cultures as follows. Putrescine (Put) concentrations increased, whereas spermidine (Spd) concentrations decreased in both free and soluble conjugated PA fractions. The concentrations of free and soluble conjugated spermine (Spm) remained low, and small amounts of excreted PAs were also found in the culture medium. The minor production of secondary metabolites reflected the undifferentiated stage of the embryogenic cell culture. Put was produced via the arginine decarboxylase (ADC) pathway. Futhermore, the gene expression data suggested that the accumulation of Put was caused neither by an increase in Put biosynthesis nor by a decrease in Put catabolism, but resulted mainly from the decrease in the biosynthesis of Spd and Spm. Put seemed to play an important role in cell proliferation in Scots pine embryogenic cells, but the low pH of the culture medium could also, at least partially, be the reason for the accumulation of endogenous Put. High Spd concentrations at the initiation of the culture, when cells were exposed to stress and cell death, suggested that Spd may act not only as a protector against stress but also as a growth suppressor, when proliferative growth is not promoted. All in all, Scots pine embryogenic cell culture was proved to be a favourable experimental platform to study PA metabolism and, furthermore, the developed system may also be beneficial in experiments where, e.g., the effect of specific stressors on PA metabolism is addressed.

Description: Many colorectal cancers (CRCs) develop in genetically susceptible individuals most of whom are not carriers of germ line mismatch repair or APC gene mutations and much of the heritable risk of CRC appears to be attributable to the co-inheritance of multiple low-risk variants. The accumulated experience to date in identifying this class of susceptibility allele has highlighted the need to conduct statistically and methodologically rigorous studies and the need for the multi-centre collaboration. This has been the motivation for establishing the COGENT (COlorectal cancer GENeTics) consortium which now includes over 20 research groups in Europe, Australia, the Americas, China and Japan actively working on CRC genetics. Here, we review the rationale for identifying low-penetrance variants for CRC and the current and future challenges for COGENT.

Description: Colorectal cancer (CRC) is one of the most common cancers worldwide with a peak of incidence in industrialised countries. It is a complex disease related to environmental and genetic risk factors. Low-penetrance genetic variations contribute significantly to sporadic and familial form of CRC. Genome-wide association studies (GWAS) have uncovered numerous robust associations between common variants and CRC risk; only a few of those were protein altering non-synonymous polymorphisms. One of the hypotheses is that non-coding and intergenic variants may change the expression levels of one or several target genes and, thus, account for a fraction of phenotypic differences, including susceptibility to CRC. Such genetic variations have been detected as expression quantitative loci (eQTLs) that show linkage/association to a large number of genes and have been defined as "master regulators of transcription". In the present work, we overview the potentialities to use results from GWAS and eQTL studies in the identification as well as investigation of master regulators in CRC susceptibility.

Description: Colorectal cancer (CRC) is a major cause of mortality throughout the world and risk of CRC is known to be modulated by nutritional factors. Low intake of the micronutrient selenium (Se) has been implicated as a risk factor in CRC, and in this article we describe the biochemical functions of selenium in selenoproteins, review the evidence for an association of selenium status with CRC and adenoma risk and describe the genetic epidemiological data on selenoprotein genes and CRC risk. Epidemiological evidence linking Se intake to CRC risk is limited but there is strong evidence for a link to adenoma risk. Two studies show an association between a genetic variant in the selenoprotein S gene and CRC risk. Selenium intake modulates selenoprotein expression in the colon, especially selenoproteins W, H, M, 15 kDa selenoprotein and glutathione peroxidase 1, and downstream targets such as endoplasmic reticulum stress response, oxidative stress and inflammatory pathways. We hypothesis that Se, through the selenoproteins, plays a key role in the ability of colonic epithelial cells to respond to microbial and oxidative challenges and that a combination of low Se intake and SNP in selenoprotein genes can impair that role and so lead to increased risk of pre-neoplastic lesions. There is a need for both further studies of selenoprotein function in the colon and major genetic epidemiological and intervention studies.

Description: Every year, approximately 1 million new colorectal cancer (CRC) cases are diagnosed and about half a million people worldwide die due to this cancer. Known differences in CRC incidence rates are mainly attributed to differences in diet and other environmental factors represented, among others, by nutrition-related complex diseases (e.g. obesity and diabetes mellitus type II). Within the last years, it has become evident that environmental risk factors can be complemented by a genetic component when considering the risk of CRC. For example, a number of polymorphisms are known to be associated with an increased risk of obesity and obesity is a risk factor for CRC. Several studies have shown that the ‘ancestral-susceptibility model’ can be reasonably applied to nutrition-related complex diseases such as obesity. The work in hand shortly discusses whether the ancestral-susceptibility model can also be applied to CRC as a nutrition-related complex disease.

Description: Alteration of DNA integrity is a potential cause of cancer and it is assumed that reduced DNA repair capacity and accumulation of DNA damage may represent intermediate markers in carcinogenesis. In this case-control study, DNA damage and nucleotide excision repair capacity (NER-DRC) were assessed in association with sporadic colorectal cancer (CRC). Both parameters were quantified by comet assay in blood cells of 70 untreated incident patients and 70 age-matched healthy controls. mRNA expression and polymorphisms in relevant NER genes were concurrently analyzed. The aim of this study was to characterize incident CRC patients for NER-DRC and to clarify possible relations between investigated variables. Comet assay and mRNA expression analysis showed that CRC patients differ in repair capacity as compared to controls. Patients had a lower NER-DRC and simultaneously they exhibited higher endogenous DNA damage (for both P 〈 0.001). Accumulation of DNA damage and decreasing NER-DRC behaved as independent modulating parameters strongly associated with CRC. Expression levels of 6 out of 9 studied genes differed between groups ( P ≤ 0.001), but none of them was related to DRC or to any of the studied NER polymorphisms. However, in patients only, XPC Ala499Val modulated expression levels of XPC , XPB and XPD gene, whereas XPC Lys939Gln was associated with XPA expression level in controls (for all P 〈 0.05). This study provides evidence on altered DRC and DNA damage levels in sporadic CRC and proposes the relevance of the NER pathway in this malignancy. Further, alterations in a complex multigene process like DNA repair may be better characterized by functional quantification of repair capacity than by quantification of individual genes transcripts or gene variants alone.

Description: APC is a key ‘gatekeeper’ gene in colorectal tumorigenesis. The high frequency of APC defects observed in colorectal cancer tissue is the result of selective growth advantage of cells with loss-of-function mutations at that locus. However, mutations may also arise due to inherent sequence instability. Defective DNA mismatch repair (MMR) and base excision repair (BER) also contribute to colorectal carcinogenesis and may compound such instability. To avoid the effect of clonal selective advantage imparted by APC mutation in cancer cells, we assessed in vitro APC mutation frequency in cell lines of lymphoid lineage to investigate the influence of defective MMR and BER. In DNA repair proficient cells, we observed substantially greater inherent sequence instability in APC gene coding sequences compared to reference sequences. Surprisingly, however, this difference was abrogated in MMR defective lines. We also found greater mutation frequency at exonic DNA sequences outwith the APC region in cells defective for either MMR or BER defects. The underlying propensity for mutation at the APC gene is intriguing, while the greater frequency of mutation in cells defective for DNA repair has relevance to understanding events leading to colorectal cancer and other malignancies.

Description: Inherited predisposition plays a role in 10–30% of colorectal cancer (CRC) cases. Of the large families with a clearly positive family history of CRC, ~40% is not affected by known CRC syndromes. The existence of families with unexplained forms of inherited CRC—familial CRC—suggests the presence of still unknown high- or moderate-risk CRC predisposing factors. While the genomic profiles of sporadic CRCs have been studied extensively, few studies have analysed the tumour profiles of hereditary or familial CRC. Here, we review recent advances in genomic tumour profiling in familial CRC in comparison with sporadic CRC. In addition, we discuss the role of known CRC risk factors in familial CRC.

Description: Despite their prime candidate status, polymorphisms near genes involved in DNA repair or in other functions related to genome stability have been conspicuously under-represented in the significant associations reported from genome-wide association studies (GWAS) of cancer susceptibility. In this study, we assessed a set of single-nucleotide polymorphisms (SNPs) near 157 DNA repair genes in three colorectal cancer (CRC) GWAS. Although no individual SNP showed evidence of association, the set of SNPs as a whole was associated with colorectal cancer risk. When candidate SNPs were examined, our data did not support most of the previously reported associations with CRC susceptibility, an exception being an effect of the MLH1 promoter SNP –93G〉A (rs1800734). Rare variants in CHEK2 (I157T and possibly del1100C) also appear to be associated with CRC risk. Overall, the absence to date of disease-associated DNA repair SNPs in cancer GWAS may be explained by a combination of the following: (i) many loci with individually very small effects on risk; (ii) rare alleles of moderate effect and (iii) subgroups of CRC, such as those with microsatellite instability, associated with specific variants. It will be particularly intriguing to determine whether any GWAS across cancer types identify DNA variants that predispose to cancers of more than one site.

Description: Over 2000 microRNA (miRNA) sequences from different species have been submitted to the miRBase, the central online repository for miRNAs, making a total of 5071 miRNA loci, expressing 5922 distinct mature miRNA sequences. In this review, we have addressed the importance of the genetic variations in humans affecting miRNAs, their target genes and the genes involved in miRNA processing for individual risk of cancer, with particular emphasis on colorectal cancer. In fact, the number of studies suggesting that individual predisposition to cancer is modulated by genetic polymorphisms affecting the biogenesis of miRNA and the interaction between miRNAs and targets has risen steeply in the last few years. We also report the first evidence that variant alleles of single-nucleotide polymorphisms (SNPs) within miRNA genes and miRNA targets, previously associated with the risk of cancer, behave differently when tested in functional studies. The SNPs belonging to the miRNA world are certainly contributing to new insights in the field of the genetic predisposition to disease.

Description: The EPICOLON consortium was initiated in 1999 by the Gastrointestinal Oncology Group of the Spanish Gastroenterology Association. It recruited consecutive, unselected, population-based colorectal cancer (CRC) cases and control subjects matched by age and gender without personal or familial history of cancer all over Spain with the main goal of gaining knowledge in Lynch syndrome and familial CRC. This epidemiological, prospective and multicentre study collected extensive clinical data and biological samples from ~2000 CRC cases and 2000 controls in Phases 1 and 2 involving 25 and 14 participating hospitals, respectively. Genetic susceptibility projects in EPICOLON have included candidate-gene approaches evaluating single-nucleotide polymorphisms/genes from the historical category (linked to CRC risk by previous studies), from human syntenic CRC susceptibility regions identified in mouse, from the CRC carcinogenesis-related pathways Wnt and BMP, from regions 9q22 and 3q22 with positive linkage in CRC families, and from the mucin gene family. This consortium has also participated actively in the identification 5 of the 16 common, low-penetrance CRC genetic variants identified so far by genome-wide association studies. Finishing their own pangenomic study and performing whole-exome sequencing in selected CRC samples are among EPICOLON future research prospects.

Description: Two forms of genomic instability can be distinguished in colorectal cancer (CRC) tumourigenesis. One is characterised by pronounced chromosomal instability (CIN), while the other relates to alterations produced at the nucleotide level that preferentially target microsatellite sequences. Tumours developing under the latter form of genomic instability possess a microsatellite instability-high (MSI-H) phenotype due to inactivation of the DNA mismatch repair system. The most recently described CRC syndrome, MUTYH -associated polyposis (MAP), shares characteristics with both MSI-H and CIN cancers. MAP carcinomas develop from the impairment of the base excision repair system, where MUTYH is involved, but also present a peculiar form of CIN. Several clinicopathological characteristics of MSI-H and MAP CRCs overlap such as tumour location, clinical prognosis and histological features. We propose that MSI-H and MAP CRCs are particularly prone to interact with their tumour microenvironment. A great deal of this interaction is probably stimulated by the immunogenic character of those tumours, known to possess a high mutagenic potential. The accumulation of mutations in coding regions of the genome of MSI-H and MAP carcinomas is likely to translate into a surplus of neo-antigens that trigger an anti-tumour immune response. The immune system constitutes thus an important vector of selective pressure that favours the outgrowth of tumour clones with immune-evasive phenotypes. In this review, we summarise the evidence for the influence of the tumour microenvironment in MSI-H and MAP tumourigenesis. Furthermore, we discuss how particular features of MSI-H and MAP CRCs can be exploited for the development of therapeutic strategies for affected patients.

Description: Colorectal cancer (CRC) is a leading cause of cancer death worldwide. Epidemiological risk factors for CRC included dietary fat intake; consequently, the role of genes in the fatty acid biosynthesis and metabolism pathways is of particular interest. Moreover, hyperlipidaemia has been associated with different type of cancer and serum lipid levels could be affected by genetic factors, including polymorphisms in the lipid metabolism pathway. The aim of this study is to assess the association between single-nucleotide polymorphisms (SNPs) in fatty acid metabolism genes, serum lipid levels, body mass index (BMI) and dietary fat intake and CRC risk; 30 SNPs from 8 candidate genes included in fatty acid biosynthesis and metabolism pathways were genotyped in 1780 CRC cases and 1864 matched controls from the Molecular Epidemiology of Colorectal Cancer study. Information on clinicopathological characteristics, lifestyle and dietary habits were also obtained. Logistic regression and association analysis were conducted. Several LIPC (lipase, hepatic) polymorphisms were found to be associated with CRC risk, although no particular haplotype was related to CRC. The SNP rs12299484 showed an association with CRC risk after Bonferroni correction. We replicate the association between the T allele of the LIPC SNP rs1800588 and higher serum high-density lipoprotein levels. Weak associations between selected polymorphism in the LIPC and PPARG genes and BMI were observed. A path analysis based on structural equation modelling showed a direct effect of LIPC gene polymorphisms on colorectal carcinogenesis as well as an indirect effect mediated through serum lipid levels. Genetic polymorphisms in the hepatic lipase gene have a potential role in colorectal carcinogenesis, perhaps though the regulation of serum lipid levels.

Description: Worldwide, colorectal cancer (CRC) is the third most common cancer, with the highest mortality rates occurring in Central Europe. The use of chemotherapy to treat CRC is limited by the inter-individual variability in drug response and the development of cancer cell resistance. ATP-binding cassette (ABC) transporters play a crucial role in the development of resistance by the efflux of anticancer agents outside of cancer cells. The aim of this study was to explore transcript levels of all human ABCs in tumours and non-neoplastic control tissues from CRC patients collected before the first line of treatment by 5-fluorouracil (5-FU)-containing regimen. The prognostic potential of ABCs was evaluated by the correlation of transcript levels with clinical factors. Relations between transcript levels of ABCs in tumours and chemotherapy efficacy were also addressed. The transcript profile of all known human ABCs was assessed using real-time polymerase chain reaction with a relative standard curve. The majority of the studied ABCs were down-regulated or unchanged between tumours and control tissues. ABCA12, ABCA13, ABCB6, ABCC1, ABCC2 and ABCE1 were up-regulated in tumours versus control tissues. Transcript levels of ABCA12, ABCC7 and ABCC8 increased in direction from colon to rectum. Additionally, transcript levels of ABCB9, ABCB11, ABCG5 and ABCG8 followed the reverse significant trend, i.e. a decrease in direction from colon to rectum. The transcript level of ABCC10 in tumours correlated with the grade ( P = 0.01). Transcript levels of ABCC6, ABCC11, ABCF1 and ABCF2 were significantly lower in non-responders to palliative chemotherapy in comparison with responders. The disease-free interval of patients treated by adjuvant chemotherapy was significantly shorter in patients with low transcript levels of ABCA7, ABCA13, ABCB4, ABCC11 and ABCD4. In conclusion, ABCC11 may be a promising candidate marker for a validation study on 5-FU therapy outcome.

Description: A functionally normal TP53 is essential to protect organisms from developing cancer. Somatic mutations in the gene represent one of the highest recurring perturbations in human tumours, including colorectal cancer (CRC). However, the variegated phenotype of wide spectrum of somatic mutations in TP53 and the complexity of the disease prevent a straight interpretation of the mutational analysis in tumours. In addition to the presence of somatic mutations, polymorphic features of the gene may also contribute to alteration of the normal TP53 functioning and variants, mainly in the form of single nucleotide polymorphisms, can be expected to impact susceptibility to sporadic CRC. In the present study, we reviewed the potential role of alterations in the TP53 gene, both somatic mutations and inherited sequence variations, in predisposition to CRC and in the prognosis and response to therapy. The available data from association studies have mostly shown contradictory outcomes. The majority of the studies were based on limited sample sizes and focussed on a limited number of polymorphisms, with main being the rs1042522 (Arg72Pro). Thus far, there is no possible generalisation of the role of TP53 as also a predictor of therapeutic response and prognosis. The effects of TP53 , and its abnormalities, on the response of tumours to cytotoxic drugs, radiation and chemoradiation are complex. However, from studies it is emerging that the inherited genetics of TP53 pathway components could be utilised to further define patient populations in their abilities to induce p53 activity in response to either DNA damaging or p53-targeted therapies.

Description: The potential of cytologically reconstructed barley line D-2946 to cope with the major lesions that hamper genome integrity, namely DNA single- and double-strand breaks was investigated. Strand breaks induced by -rays and Li ions were assessed by neutral and alkaline comet assay. Repair capacity after bleomycin treatment was evaluated by agarose gel electrophoresis under neutral and alkaline conditions. Frequencies of radiation-induced chromosome aberrations were also determined. Results indicate that radiation-mediated constitutive rearrangement of the chromosome complement has led to a substantial modulation of the sensitivity of barley genome towards DNA strand breaks, produced by ionising radiation, Li ion implantation and bleomycin in an agent-specific manner, as well as of the clastogenic response to -rays. Based on these findings, reconstructed barley karyotype D-2946 can be considered a candidate radio-sensitive line with reduced ability to maintain genome integrity with respect to both DNA and chromosomal damage.

Description: The trace element selenium is an essential micronutrient that has received considerable attention for its potential use in the prevention of cancer. In spite of this interest, the mechanism(s) by which selenium might function as a chemopreventive remain to be determined. Considerable experimental evidence indicates that one possible mechanism by which selenium supplementation may exert its benefits is by enhancing the DNA damage repair response, and this includes data obtained using cultured cells, animal models as well as in human clinical studies. In these studies, selenium supplementation has been shown to be beneficial in reducing the frequency of DNA adducts and chromosome breaks, consequentially reducing the likelihood of detrimental mutations that ultimately contribute to carcinogenesis. The benefits of selenium can be envisioned as being due, at least in part, to it being a critical constituent of selenoproteins such as glutathione peroxidases and thioredoxin reductases, proteins that play important roles in antioxidant defence and maintaining the cellular reducing environment. Selenium, therefore, may be protective by preventing DNA damage from occurring as well as by increasing the activity of repair enzymes such as DNA glycosylases and DNA damage repair pathways that involve p53, BRCA1 and Gadd45. An improved understanding of the mechanism of selenium’s impact on DNA repair processes may help to resolve the apparently contradicting data obtained from decades of animal work, human epidemiology and more recently, clinical supplementation studies.

Description: The tumour-suppressor gene BRCA2 has been demonstrated to be involved in maintenance of genome integrity by affecting DNA double-strand break repair and homologous recombination. Protein-truncating mutations in BRCA2 predispose women to early onset breast and ovarian cancers and account for 15–30% of familial breast cancer risk. In contrast, the human cancer risk due to missense mutations, intronic variants, and in-frame deletions and insertions in the BRCA2 gene, called unclassified variants, has not been determined. Here, we want to define if the yeast Saccharomyces cerevisiae is a good model to study the role of BRCA2 in DNA recombination and repair and to characterise the unclassified BRCA2 missense variants. Therefore, we expressed the wild-type BRCA2 in yeast and determined the effect of BRCA2 on yeast homologous recombination, methyl methanesulphonate (MMS)-induced Rad51 and Rad52 foci and MMS sensitivity. The expression of BRCA2 induces a high increase in both intra- and inter-recombination events and confers a higher MMS resistance as compared with the negative control. This may suggest that BRCA2 gets involved in DNA repair pathways in yeast. Moreover, the expression of BRCA2 did not affect the number of cells carrying Rad51 or Rad52 nuclear foci. Finally, we aimed to investigate if yeast could be reliable system to set up a functional assay to distinguish a mutated protein from a neutral polymorphism. Therefore, we have expressed two neutral (M1915T and A2951T) and one pathogenic variant (G2748D) in yeast and checked the effect on recombination. The neutral M1915T variant increased intra-chromosomal recombination by almost 2-fold and the other neutral A2975T variant increased intra-chromosomal recombination 2.5-fold as compared with the control. On the other end, the pathogenic variant G2748D did not increase intra- and inter-chromosomal recombination in yeast and, consequently, confers a phenotype very different from the wild-type BRCA2. Moreover, we have also evaluated whether the expression of the selected unclassified variants affects homologous recombination in yeast. Results indicated that the expression of the selected BRCA2 variants differentially affects yeast recombination suggesting that yeast could be a very promising genetic system to characterise BRCA2 missense variants.

Description: Alcohol consumption is frequently associated with various cancers and the enhancement of the metabolic activation of carcinogens has been proposed as a mechanism underlying this relationship. The ethanol-induced enhancement of N -nitrosodiethylamine (DEN)-mediated carcinogenesis can be attributed to an increase in hepatic activity. However, the mechanism of elevation of N -nitrosomethylbenzylamine (NMBA)-induced tumorigenesis remains unclear. To elucidate the mechanism underlying the role of ethanol in the enhancement of NMBA-induced oesophageal carcinogenesis, we evaluated the hepatic and extrahepatic levels of the cytochrome P450 (CYP) and mutagenic activation of environmental carcinogens by immunoblot analyses and Ames preincubation test, respectively, in F344 rats treated with ethanol. Five weeks of treatment with 10% ethanol added to the drinking water or two intragastric treatments with 50% ethanol, both resulted in elevated levels of CYP2E1 (1.5- to 2.3-fold) and mutagenic activities of DEN, N -nitrosodimethylamine and N -nitrosopyrrolidine in the presence of rat liver S9 (1.5- to 2.4-fold). This was not the case with CYP1A1/2, CYP2A1/2, CYP2B1/2 or CYP3A2, nor with the activities of 2-amino-3-methylimidazo[4,5- f ]quinoline, 3-amino-1-methyl-5 H -pyrido[4,3- b ]indole, aflatoxin B 1 or other N -nitroso compounds (NOCs), including NMBA. Ethanol-induced elevations of CYP2A and CYP2E1 were observed in the oesophagus (up to 1.7- and 2.3-fold) and kidney (up to 1.5- and 1.8-fold), but not in the lung or colon. In oesophagus and kidney, the mutagenic activities of NMBA and four NOCs were markedly increased (1.3- to 2.4-fold) in treated rats. The application of several CYP inhibitors revealed that CYP2A were likely to contribute to the enhancing effect of ethanol on NMBA activation in the rat oesophagus and kidney, but that CYP2E1 failed to do so. These results showed that the enhancing effect of ethanol on NMBA-induced oesophageal carcinogenesis could be attributed to an increase in the metabolic activation of NMBA by oesophageal CYP2A during the initiation phase, and that this occurred independently of CYP2E1.

Description: The comet assay is increasingly used to measure the repair of various types of DNA damage. Modifications of the standard protocol have been introduced to determine the repair capacity of specific DNA repair pathways by the removal of pathway-specific DNA lesions. Recently, a cellular phenotype assay for nucleotide excision repair (NER) by quantifying the DNA strand breaks after in vitro challenge of peripheral blood mononucleated cells with benzo[ a ]pyrene diol epoxide (BPDE) in the presence or absence of the DNA polymerase inhibitor aphidicolin (APC) was developed (Vande Loock, K., Decordier, I., Ciardelli, R., Haumont, D. and Kirsch-Volders, M. (2010) An aphidicolin-block nucleotide excision repair assay measuring DNA incision and repair capacity. Mutagenesis , 25, 25–32). Individual repair capacity (RC) was defined as the amount of DNA damage induced by BPDE in the presence of APC minus the damage induced by BPDE and APC alone. This value should mainly reflect the incision capacity of the NER enzymes. Following this approach, we investigated the RC of cultured isolated peripheral blood mononuclear cells of nine donors in repeated experiments. We also performed the same experiments with peripheral whole blood cultures from these donors. Our results indicated considerable intra- and inter-individual variability and substantial differences between the RC of isolated mononuclear cells and whole blood from the same donor. Furthermore, the RC of unstimulated blood did not differ significantly from the repair capacity of stimulated blood but also showed considerable inter-individual variability. Altogether, our results suggest that there is still need for standardisation and validation of this assay before it can be reliably used in human biomonitoring studies.

Description: Genetic polymorphisms can partially explain the large inter-individual variation in DNA adduct levels following exposure to polycyclic aromatic hydrocarbons. Effects of genetic polymorphisms on DNA adduct formation are difficult to assess in human studies because exposure misclassification attenuates underlying relationships. Conversely, ex vivo studies offer the advantage of controlled exposure settings, allowing the possibility to better elucidate genotype–phenotype relationships and gene–gene interactions. Therefore, we exposed lymphocytes of 168 non-smoking volunteers ex vivo to the environmental pollutant benzo(a)pyrene (BaP) and BaP-related DNA adducts were quantified. Thirty-four genetic polymorphisms were assessed in genes involved in carcinogen metabolism, oxidative stress and DNA repair. Polymorphisms in catalase ( CAT , rs1001179) and cytochrome P450 1B1 ( CYP1B1 , rs1800440) were significantly associated with DNA adduct levels, especially when combined. Moreover, reverse transcription–polymerase chain reaction (RT–PCR) analysis in a subset of 30 subjects revealed that expression of catalase correlated strongly with expression of CYP1B1 ( R = 0.92, P 〈 0.001). To further investigate the mechanism by which catalase influences CYP1B1 and how they simultaneously affect BaP-related DNA adduct levels, catalase expression was transiently knocked down in the human lung epithelial cell line A549. Although catalase knockdown did not immediately change CYP1B1 gene expression, recovery of catalase expression 8 h after the knockdown coincided with a 2.2-fold increased expression of CYP1B1 ( P 〈 0.05). We conclude that the genetic polymorphism in the promoter region of CAT may determine the amount and activity of catalase, which may subsequently regulate the expression of CYP1B1. As a result, both genetic polymorphisms modulate DNA adduct levels in lymphocytes by BaP ex vivo .

Description: The aetiology of head and neck cancer (HNC) has been shown to be associated with genetic and certain environmental factors that produce DNA damage. Base excision repair (BER) genes are responsible for repair of DNA damage caused by reactive oxygen species and other electrophiles and therefore are good candidate susceptibility genes for HNC. Apurinic/apyrimidinic endonuclease-1 ( APEX1 ) proteins have important functions in the BER pathway. In this case–control study, all exons of the APEX1 gene and its exon/intron boundaries were amplified in 300 HNC cases and 300 matched healthy controls and then analysed by single-stranded conformational polymorphism. Amplified products showing altered mobility patterns were sequenced and analysed. To confirm our observations, we examined APEX1 expression at mRNA level on 50 head and neck squamous cell carcinoma (HNSCC) and 50 normal control samples by quantitative real-time polymerase chain reaction. At germ line level, three novel mutations (13T 〉 G, Ser129Arg and Val131Gly) of APEX1 were observed. The homozygous and heterozygous genotypes of APEX1 13T 〉 G, Ser129Arg and Val131Gly appear to be significantly involved in the development of HNC. In the case of expressional level, APEX1 mRNA expression was positively correlated with tumour size, clinical stage and positive lymph node metastasis. Statistical analysis showed a significantly higher APEX1 mRNA level in HNC tumour tissue than in control samples. Our study demonstrated that APEX1 mutations and deregulation of APEX1 are associated with increased risk of HNC in the Pakistani population.

Description: Patients with chronic kidney disease (CKD) have signs of genomic instability and, as a consequence, extensive genetic damage, possibly due to accumulation of uraemic toxins, oxidative stress mediators and other endogenous substances with genotoxic properties. We explored factors associated with the presence and background levels of genetic damage in CKD. A cross-sectional study was performed in 91 CKD patients including pre-dialysis (CKD patients; n = 23) and patients undergoing peritoneal dialysis (PD; n = 33) or haemodialysis (HD; n = 35) and with 61 healthy subjects, divided into two subgroups with the older group being in the age range of the patients, serving as controls. Alkaline comet assay and cytokinesis-block micronucleus assay in peripheral blood lymphocytes were used to determine DNA and chromosome damage, respectively, present in CKD. Markers of oxidative stress [malondialdehyde (MDA), advanced glycation end products (AGEs), thiols, advanced oxidation protein products and 8-hydroxy-2'-deoxyguanosine] and markers of inflammation (C-reactive protein, interleukin-6 and tumour necrosis factor alpha) were also measured. Micronucleus (MN) frequency was significantly higher ( P 〈 0.05) in the CKD group (46±4) when compared with the older control (oC) group (27.7±14). A significant increase in MN frequency ( P 〈 0.05) was also seen in PD patients (41.9±14) versus the oC group. There was no statistically significant difference for the HD group (29.7±15.6; P = NS) versus the oC group. Comet assay data showed a significant increase ( P 〈 0.001) of tail DNA intensity in cells of patients with CKD (15.6±7%) with respect to the total control (TC) group (11±1%). PD patients (14.8±7%) also have a significant increase ( P 〈 0.001) versus the TC group. Again, there was no statistically significant difference for the HD group (12.5±3%) compared with the TC group. Patients with MN values in the upper quartile had increased cholesterol, triglycerides, AGEs and MDA levels and lower albumin levels. Multiple logistic regression analysis showed that male gender, diabetes and treatment modality were independently associated with higher levels of DNA damage. Our results suggest that oxidative stress, diabetes, gender and dialysis modality in CKD patients increased DNA and chromosome damage. To confirm these data, prospective clinical trials need to be performed.

Description: Recent studies suggest increased cancer risk in patients with type 2 diabetes mellitus (T2DM) compared with healthy individuals. The present study aims to assess whether T2DM is associated with increased genome instability and whether a healthy diet with natural foods can improve genome stability in peripheral blood lymphocytes (PBLs). Seventy-six diabetic and 21 non-diabetic individuals were randomly assigned to either an ‘intervention’ or an ‘information only’ group. All participants received information about the beneficial effects of a healthy diet, while subjects of the intervention group received additionally 300g of vegetables and 25ml of plant oil rich in polyunsaturated fatty acids per day for 8 weeks. Chromosomal damage was assessed using the cytokinesis-block micronucleus (MN) cytome assay. Levels of chromosomal damage did not differ between diabetic and non-diabetic individuals. However, diabetic individuals with MN frequency above the high 50th percentile had significantly higher levels of fasting plasma glucose, glycosylated haemoglobin and were at higher risk for cardiovascular disease (CVD), assessed by the Framingham general cardiovascular risk score. Non-diabetic individuals with MN frequency above the 50th percentile had significantly lower vitamin B12 levels. The intervention with vegetables and plant oil led to significant increases in folate, -tocopherol, α- and β-carotene while vitamin B12 was significantly reduced. Levels of chromosomal damage were not altered, only apoptosis was slightly increased. The results suggest interactions between glycaemic control, CVD risk and genome stability in individuals with T2DM. However, a healthy diet does not improve genome damage in PBLs.

Description: A novel selective glucocorticoid receptor (GR) agonist, AZD2906, was found to increase the incidence of micronucleated immature erythrocytes (MIE) in the bone marrow of rats given two oral doses at the maximum tolerated level. Because GR agonists as a class are considered not to be genotoxic and AZD2906 showed no activity in the standard in vitro tests or in vivo in a rat liver comet assay, investigative studies were performed to compare AZD2906 with a reference traditional GR agonist, prednisolone. Emphasis was placed on blood and bone marrow parameters in these studies because GR activation has been reported to induce erythropoiesis which, in turn, is known to increase MIE in the bone marrow. Both compounds induced almost identical, small increases in micronucleus frequency at all doses tested. Directly comparable changes in haematological and bone marrow parameters were also seen with significant decreases in lymphoid cells in both compartments and significant increases in numbers of circulating neutrophils. Although no evidence of increased erythropoiesis was seen as increased immature erythrocyte numbers either in the blood or in the bone marrow, histopathological examination showed focal areas in the bone marrow where the erythroid population was enriched in association with an atrophic myeloid lineage. This could have been due to direct stimulation of the erythroid lineage or a secondary effect of myelosuppression inducing a rebound increase in erythropoiesis into the vacant haematopoietic cell compartment. It was concluded that the increased MIE frequencies induced by both AZD2906 and prednisolone are a consequence of their pharmacological effects on the bone marrow, either by directly inducing erythropoiesis or by some other unknown effect on cellular function, and do not indicate potential genotoxicity. This conclusion is supported by the lack of carcinogenic risk in man demonstrated by decades of clinical use of prednisolone and other GR agonists.

Description: Although amorphous silica is used in food products, cosmetics and paints and as vector for drug delivery, data on its potential health hazard are limited. The aim of this study was to investigate the cytotoxic and genotoxic potential of silica particles of different sizes (250 and 500nm) and structures (dense and mesoporous). Dense silica (DS) spheres were prepared by sol–gel synthesis, mesoporous silica particles (MCM-41) were prepared using hexadecyltrimethyl ammonium bromide as a structure-directing agent and tetraethylorthosilicate as silica source. Particles were accurately characterised by dynamic light scattering, nitrogen adsorption, X-ray diffraction and field emission scanning electron microscopy. Murine macrophages (RAW264.7) and human epithelial lung (A549) cell lines were selected for investigation. Genotoxicity was evaluated by Comet assay and micronucleus test. Cytotoxicity was tested by the trypan blue method. Cells were treated with 0, 5, 10, 20, 40 and 80 µg/cm 2 of different silica powders for 4 and 24 h. The intracellular localisation of silica was investigated by transmission electron microscopy. Amorphous particles penetrated into the cells, being compartmentalised within endocytic vacuoles. DS and MCM-41 particles induced cytotoxic and genotoxic effects in A549 and RAW264.7 although to different extent in the two cell lines. A549 were resistant in terms of cell viability, but showed a generalised induction of DNA strand breaks. RAW264.7 were susceptible to amorphous silica exposure, exhibiting both cytotoxic and genotoxic responses as DNA strand breaks and chromosomal alterations. The cytotoxic response of RAW264.7 was particularly relevant after MCM-41 exposure. The genotoxicity of amorphous silica highlights the need for a proper assessment of its potential hazard for human health.

Description: Previous studies in twins indicate that non-shared environment, beyond genetic factors, contributes substantially to individual variation in mutagen sensitivity; however, the role of specific causative factors (e.g. tobacco smoke, diet) was not elucidated. In this investigation, a population of 22 couples of monozygotic twins with discordant smoking habits was selected with the aim of evaluating the influence of tobacco smoke on individual response to DNA damage. The study design virtually eliminated the contribution of genetic heterogeneity to the intra-pair variation in DNA damage response, and thus any difference in the end-points investigated could directly be attributed to the non-shared environment experienced by co-twins, which included as main factor cigarette smoke exposure. Peripheral lymphocytes of study subjects were challenged ex vivo with -rays, and the induction, processing, fixation of DNA damage evaluated through multiple approaches. Folate status of study subjects was considered significant covariate since it is affected by smoking habits and can influence radiosensitivity. Similar responses were elicited by -rays in co-twins for all the end-points analysed, despite their discordant smoking habits. Folate status did not modify DNA damage response, even though a combined effect of smoking habits, low-plasma folic acid level, and ionising radiation was observed on apoptosis. A possible modulation of DNA damage response by duration and intensity of tobacco smoke exposure was suggested by Comet assay and micronucleus data, but the effect was quantitatively limited. Overall, the results obtained indicate that differences in smoking habits do not contribute to a large extent to inter-individual variability in the response to radiation-induced DNA damage observed in healthy human populations.

Description: AZD9708 is a new chemical entity with selective and long-acting β2-agonistic properties currently being evaluated by AstraZeneca for potential use in treatment of respiratory diseases by the inhaled route. As part of the toxicological characterisation of this compound, an increased incidence of micronucleated immature erythrocytes (MIEs) was seen in the bone marrow of rats following single intravenous doses near the maximum tolerated. This effect was seen in the absence of in vitro genotoxicity in bacterial and mammalian cells and no consistent evidence of in vivo DNA damage in the the bone marrow or liver using the comet assay was observed. Because of the lack of signals for mutagenic potential, combined with the observation that MIE frequencies appeared to be increased in only some of the rats and the clearest response was seen at the intermediate dose, it was hypothesised that the effect was secondary to β2-adrenergic receptor overstimulation. Because it appears that this has not been previously described for β2-agonists and because pharmacodynamic/pharmacokinetic factors may influence the response, studies using repeated dosing were performed to investigate whether this would lead to compound-induced tachyphylaxis with tolerance induction and decreased responses indicated by β2-effect biomarkers. A series of experiments confirmed that a sequence of five escalating daily doses leading to systemic exposure corresponding to that after a single dose led to symptomatic tolerance, declining or diminished effects on plasma biomarkers of β2-effects (plasma glucose and potassium) and elimination of the micronucleus response. This suggests that the increased MIE frequencies after single doses of AZD9708 are secondary to physiological overstimulation of β2-adrenergic receptors, not a consequence of genotoxicity.

Description: In human skin, the 3895-bp deletion of mitochondrial DNA (mtDNA 3895 ) is catalysed by ultraviolet (UV) light through the generation of reactive oxygen species. Given its function in vision, the human eye is exposed to oxidising UV and blue light in its anterior (cornea, iris) and posterior (retina) structures. In this study, we employed a highly sensitive quantitative PCR technique to determine mtDNA 3895 occurrence in human eye. Our analysis shows that the mtDNA 3895 is concentrated in both the cornea and the retina. Within the cornea, the highest mtDNA 3895 level is found in the stroma, the cellular layer conferring transparency and rigidity to the human cornea. Moreover, mtDNA 3895 accumulates with age in the stroma, suggesting a role of this deletion in corneal ageing. Within the retina, mtDNA 3895 is concentrated in the macular region of both the neural retina and the retinal pigment epithelium, supporting the hypothesis that this deletion is implicated in retinal pathologies such as age-related macular degenerescence. Taken together, our results imply that UV and blue light catalyse mtDNA 3895 induction in the human eye.

Description: Although there are several in vivo tests for potential genotoxicity, with the possible exception of the transgenic rodent mutation models, none is specifically intended to assess increasing damage with chronic administration. In principle, peripheral blood lymphocytes would be expected to accumulate DNA damage with repeated dosing because the majority are not in active division and appear to have limited DNA repair capability, and they are exposed to plasma levels of test materials and metabolites. However, there appear to be no published reports confirming this principle. Therefore, in the current study, after optimising culture conditions for rat lymphocytes in this laboratory, rats were given oral doses of cyclophosphamide or hexamethylphosphoramide (HMPA) for up to 28 days and peripheral lymphocytes analysed for chromosome aberrations at various time points. The results clearly show that, for both compounds, doses that gave no significant increases in aberration frequency after 2 days induced clear increases after 15 days with further damage detectable after 28 doses. With HMPA, it was shown that DNA damage persisted for at least 10 days after cessation of treatment. These data show that repeat dose studies in the rat measuring chromosome aberration frequency in lymphocytes can give a genuine indication that genotoxicity may increase with chronic administration and, therefore, maybe useful in assessing the risk of potentially genotoxic substances.

Description: The parasitic disease human African trypanomiasis (HAT), also known as sleeping sickness, is a highly neglected fatal condition endemic in sub-Saharan Africa, which is poorly treated with medicines that are toxic, no longer effective or very difficult to administer. New, safe, effective and easy-to-use treatments are urgently needed. Many nitroimidazoles possess antibacterial and antiprotozoal activity and examples such as tinidazole are used to treat trichomoniasis and guardiasis, but concerns about toxicity including genotoxicity limit their usefulness. Fexinidazole, a 2-substituted 5-nitroimidazole rediscovered by the Drugs for Neglected Diseases initiative (DNDi) after extensive compound mining of public and pharmaceutical company databases, has the potential to become a short-course, safe and effective oral treatment, curing both acute and chronic HAT. This paper describes the genotoxicity profile of fexinidazole and its two active metabolites, the sulfoxide and sulfone derivatives. All the three compounds are mutagenic in the Salmonella /Ames test; however, mutagenicity is either attenuated or lost in Ames Salmonella strains that lack one or more nitroreductase(s). It is known that these enzymes can nitroreduce compounds with low redox potentials, whereas their mammalian cell counterparts cannot, under normal conditions. Fexinidazole and its metabolites have low redox potentials and all mammalian cell assays to detect genetic toxicity, conducted for this study either in vitro (micronucleus test in human lymphocytes) or in vivo ( ex vivo unscheduled DNA synthesis in rats; bone marrow micronucleus test in mice), were negative. Thus, fexinidazole does not pose a genotoxic hazard to patients and represents a promising drug candidate for HAT. Fexinidazole is expected to enter Phase II clinical trials in 2012.

Description: The mouse liver tumorigenic conazole fungicides triadimefon and propiconazole have previously been shown to be in vivo mouse liver mutagens in the Big Blue™ transgenic mutation assay when administered in feed at tumorigenic doses, whereas the nontumorigenic conazole myclobutanil was not mutagenic. DNA sequencing of the mutants recovered from each treatment group as well as from animals receiving control diet revealed that propiconazole- and triadimefon-induced mutations do not represent general clonal expansion of background mutations, and support the hypothesis that they arise from the accumulation of endogenous reactive metabolic intermediates within the liver in vivo . We therefore measured the spectra of endogenous DNA adducts in the livers of mice from these studies to determine if there were quantitative or qualitative differences between mice receiving tumorigenic or nontumorigenic conazoles compared to concurrent control animals. We resolved and quantitated 16 individual adduct spots by 32 P postlabelling and thin layer chromatography using three solvent systems. Qualitatively, we observed the same DNA adducts in control mice as in mice receiving conazoles. However, the 13 adducts with the highest chromatographic mobility were, as a group, present at significantly higher amounts in the livers of mice treated with propiconazole and triadimefon than in their concurrent controls, whereas this same group of DNA adducts in the myclobutanil-treated mice was not different from controls. This same group of endogenous adducts were significantly correlated with mutant frequency across all treatment groups ( P = 0.002), as were total endogenous DNA adduct levels ( P = 0.005). We hypothesise that this treatment-related increase in endogenous DNA adducts, together with concomitant increases in cell proliferation previously reported to be induced by conazoles, explain the observed increased in vivo mutation frequencies previously reported to be induced by treatment with propiconazole and triadimefon.

Description: The comet assay or single cell gel electrophoresis has proven to be a versatile and sensitive method of measuring the induction and repair of DNA damage in individual cells. However, one of the drawbacks of the assay is the bias caused by changes in the ability of cells to repair DNA damage in different cell cycle phases. Whereas the bias seems less important when G0 peripheral blood lymphocytes are studied, it might cause problems when proliferating cells are investigated. In this paper, we validate the assumption that the total comet fluorescence intensity corresponds to the position of the cell in the cell cycle and can be used to assign single cells to specific cell cycle phases. To validate the approach, we used a very homogenous blood mononuclear CD34 + cell population in G0 phase (unstimulated) or stimulated to enter the cell cycle. An analysis of the cell cycle distribution revealed that the 15 comet intensity classes and the 100 comets usually analyzed in a typical comet experiment are sufficient to obtain a reliable cell cycle distribution comparable with the results obtained by the flow cytometry for the same cell population. The effect of the cell cycle position on the results obtained by the comet assay for proliferating and non-proliferating cell populations irradiated with 3 Gy of X-radiation is also discussed.

Description: Disruptions of normal apoptotic pathways, which are mainly mediated by caspases, play an essential role in cancer development. Caspase-8 (CASP8) is encoded by the CASP8 gene and is centrally involved in the apoptosis of T lymphocytes. The association between a six-nucleotide deletion polymorphism (-652 6N del) of the CASP8 gene and the risk of cancer is widely reported; however, study results have been inconsistent and contradictory. To evaluate the association between the CASP8 -652 6N del polymorphism and the risk of cancer and to overcome the limitations of any individual study, a meta-analysis based on a total of 23 700 cases and 26 412 controls from 30 case–control studies was conducted. The results of the overall analysis suggested that the CASP8 -652 6N del polymorphism is associated with decreased risk of cancer for the allele contrast [del versus ins: odd ratio (OR) = 0.86, 95% confidence interval (CI) = 0.80–0.92], the additive genetic model (del/del versus ins/ins: OR = 0.78, 95% CI = 0.69–0.88), the dominant genetic model (del/del+del/ins versus ins/ins: OR = 0.83, 95% CI = 0.78–0.89) and the recessive genetic model (del/del versus ins/ins+del/ins: OR = 0.84, 95% CI = 0.75–0.93). In addition, after stratification for ethnicity and cancer type, significantly reduced risk was found for Asians and Caucasians as well as for individuals in the colorectal cancer group and the ‘other cancers’ group. Accordingly, there is an association between the CASP8 -652 6N del polymorphism and reduced cancer risk, especially among Asians, Caucasians and those with colorectal cancer. However, further research, such as studies focusing on additional ethnic groups and cancer types, is needed to provide a more exact and comprehensive synthesis conclusion.

Description: Increased serum bile salt levels have been associated to a single-nucleotide polymorphism in the bile salt export pump (BSEP; ABCB11 ) in several acquired cholestatic liver diseases but there is little evidence in alcoholic liver disease (ALD). Furthermore, a crosstalk between vitamin D and bile acid synthesis has recently been discovered. Whether this crosstalk has an influence on the course of ALD is unclear to date. Our aim was to analyse the role of genetic polymorphisms in BSEP and the vitamin D receptor gene ( NR1I1 ) on the emergence of cirrhosis in patients with ALD. Therefore, 511 alcoholic patients (131 with cirrhosis and 380 without cirrhosis) underwent ABCB11 genotyping (rs2287622). Of these, 321 (131 with cirrhosis and 190 without cirrhosis) were also tested for NR1I1 polymorphisms (bat-haplotype: BsmI rs1544410, ApaI rs7975232 and TaqI rs731236). Frequencies of ABCB11 and NR1I1 genotypes and haplotypes were compared between alcoholic patients with and without cirrhosis and correlated to serum bile salt, bilirubin and aspartate aminotransferase levels in those with cirrhosis. Frequencies of ABCB11 and NR1I1 genotypes and haplotypes did not differ between the two subgroups and no significant association between genotypes/haplotypes and liver function tests could be determined for neither polymorphism. We conclude that ABCB11 and NR1I1 polymorphisms are obviously not associated with development of cirrhosis in patients with ALD.

Description: Newborns have to cope with hypoxia during delivery and a sudden increase in oxygen at birth. Oxygen will partly be released as reactive oxygen species having the potential to cause damage to DNA and proteins. In utero , increase of most (non)-enzymatic antioxidants occurs during last weeks of gestation, making preterm neonates probably more sensitive to oxidative stress. Moreover, it has been hypothesized that oxidative stress might be the common etiological factor for certain neonatal diseases in preterm infants. The aim of this study was to assess background DNA damage; in vitro H 2 O 2 induced oxidative DNA damage and repair capacity (residual DNA damage) in peripheral blood mononucleated cells from 25 preterm newborns and their mothers. In addition, demographic data were taken into account and repair capacity of preterm was compared with full-term newborns. Multivariate linear regression analysis revealed that preterm infants from smoking fathers have higher background DNA damage levels than those from non-smoking fathers, emphasizing the risk of paternal smoking behaviour for the progeny. Significantly higher residual DNA damage found after 15-min repair in preterm children compared to their mothers and higher residual DNA damage after 2 h compared to full-term newborns suggest a slower DNA repair capacity in preterm children. In comparison with preterm infants born by caesarean delivery, preterm infants born by vaginal delivery do repair more slowly the in vitro induced oxidative DNA damage. Final impact of passive smoking and of the slower DNA repair activity of preterm infants need to be confirmed in a larger study population combining transgenerational genetic and/or epigenetic effects, antioxidant levels, genotypes, repair enzyme efficiency/levels and infant morbidity.

Description: Experimental evidences suggest that most essential oils possess a wide range of biological and pharmacological activities that may protect tissues against oxidative damage. In this study, we investigated DNA-protective effect of borneol, a component of many essential oils, against oxidative DNA damage induced in primary cultures of rat hepatocytes. Borneol was added to drinking water of Sprague–Dawley rats and DNA resistance against oxidative agents was compared in hepatocytes originated from control and borneol-treated rats. Oxidative stress induced by visible light-excited methylene blue (MB/VL) or 2,3-dimethoxy-1,4-naphthoquionone (DMNQ) resulted in increased levels of DNA lesions measured by the modified single cell gel electrophoresis. Borneol (17 or 34 mg/kg body weight) added to drinking water of rats for 7 days reduced the level of oxidative DNA lesions induced in their hepatocytes by MB/VL or DMNQ. To explain the increased resistance of DNA towards oxidative stress, we measured the base-excision repair (BER) capacity in liver cell extracts of control and borneol-supplemented rats on DNA substrate of HepG2 cells containing oxidative damage. Our results showed that administration of borneol in drinking water had no effect on incision activity of hepatocytes isolated from supplemented rats. The spectrophotometric assessment of enzymatic antioxidants superoxide dismutase (SOD) and glutathione peroxidase (GPx) activities and the flow cytometric assessment of total intracellular glutathione (iGSH) in primary hepatocytes of borneol-supplemented rats showed no changes in SOD and GPx activities but higher iGSH content particularly in hepatocytes of higher borneol dose (34 mg/kg) supplemented rats in comparison to control animals. Despite the fact that borneol had no effect either on BER of oxidative DNA damage or on the levels of antioxidant enzymes and manifested no reducing power and radicals scavenging activity, it increased significantly the level of non-enzymatic antioxidant iGSH which could reduce the oxidative DNA lesions induced by MB/VL or DMNQ.

Description: Several heat-based sap flow methods, such as the heat field deformation method and the heat ratio method, include the thermal diffusivity D of the sapwood as a crucial parameter. Despite its importance, little attention has been paid to determine D in a plant physiological context. Therefore, D is mostly set as a constant, calculated during zero flow conditions or from a method of mixtures, taking into account wood density and moisture content. In this latter method, however, the meaning of the moisture content is misinterpreted, making it theoretically incorrect for D calculations in sapwood. A correction to this method, which includes the correct application of the moisture content, is proposed. This correction was tested for European and American beech and Eucalyptus caliginosa Blakely & McKie. Depending on the dry wood density and moisture content, the original approach over- or underestimates D and, hence, sap flux density by 10% and more.

Description: The evergreen holm oak Quercus ilex L. is the most representative tree in Mediterranean forests. Accurate estimation of the limiting factors of photosynthesis for Q. ilex and the prediction of ecosystem water-use efficiency by mechanistic models can be achieved only by establishing whether this species shows heterogenic stomatal aperture, and, if so, the circumstances in which this occurs. Here, we collected gas-exchange and chlorophyll fluorescence data in Q. ilex leaves from a nursery to measure the effects of stomatal oscillations on PSII quantum yield ( PSII ) under water stress. Stomatal conductance ( g s ) was used as an integrative indicator of the degree of water stress. Images of chlorophyll fluorescence showed heterogeneous PSII when g s was 〈50 mmol H 2 O m –2 s –1 , representative of severe drought and corresponding to a container capacity 〈45%. Stomatal patchiness was related to a coefficient of variation (CV) of PSII values 〉2.5%. A parallel study in the forest confirmed heterogeneous PSII values in leaves in response to declining water availability. Three kinds of Q. ilex individuals were distinguished: those resprouting after a clear-cut (resprouts, R); intact individuals growing in the same clear-cut area as resprouts (controls, C); and intact individuals in a nearby, undisturbed area (forest controls, CF). Patchiness increased in C and CF in response to increasing drought from early May to late July, whereas in R, PSII values were maintained as a result of their improved water relations since the pre-existing roots were associated with a smaller aerial biomass. Patchiness was related to a % CV of PSII values 〉4 and associated in the summer with mean g s values of 30 mmol H 2 O m –2 s –1 . Under milder drought in spring, PSII patchiness was less strictly related to g s variations, pointing to biochemical limitants of photosynthesis. The occurrence of heterogenic photosynthesis caused by patchy stomatal closure in Q. ilex during severe drought should be taken into account in ecosystem modelling in which harsher water stress conditions associated with climate change are predicted.

Description: The freezing pattern and frost killing temperatures of apple ( Malus domestica Borkh.) xylem were determined by differential thermal analysis and infrared differential thermal analysis (IDTA). Results from detached or attached twigs in controlled freezing experiments and during natural field freezing of trees were compared. Non-lethal freezing of apoplastic water in apple xylem as monitored during natural winter frosts in the field occurred at –1.9 ± 0.4 °C and did not change seasonally. The pattern of whole tree freezing was variable and specific to the environmental conditions. On detached twigs high-temperature freezing exotherms (HTEs) occurred 2.8 K below the temperature observed under natural frosts in the field with a seasonal mean of –4.7 ± 0.5 °C. Microporous apple xylem showed freezing without a specific pattern within a few seconds in IDTA images during HTEs, which is in contrast to macroporous xylem where a 2D freezing pattern mirrors anatomical structures. The pith tissue always remained unfrozen. Increasing twig length increased ice nucleation temperature; for increased twig diameter the effect was not significant. In attached twigs frozen in field portable freezing chambers, HTEs were recorded at a similar mean temperature (–4.6 ± 1.0 °C) to those for detached twigs. Upon lethal intracellular freezing of apple xylem parenchyma cells (XPCs) low-temperature freezing exotherms (LTEs) can be recorded. Low-temperature freezing exotherms determined on detached twigs varied significantly between a winter minimum of –36.9 °C and a summer maximum –12.7 °C. Within the temperature range wherein LTEs were recorded by IDTA in summer (–12.7 ± 0.5 to –20.3 ± 1.1 °C) various tiny clearly separated discontinuous freezing events could be detected similar to that in other species with contrasting XPC anatomy. These freezing events appeared to be initially located in the primary and only later in the secondary xylem. During the LTE no freezing events in the bark and central pith tissue were recorded. Attached twigs were exposed to various freezing temperatures at which LTEs occur. Even if 60% of XPCs were frost-damaged twigs were able to recuperate and showed full re-growth indicating a high regeneration capacity even after severe frost damage to XPCs.

Description: Nitrogen (N) retranslocation within tree canopies has been intensively studied and assumed to function as a one-way process (e.g., from older to newer leaves). However, recent studies have found that both N output and input occur in individual leaves, suggesting that ‘gross’ N retranslocation exists behind ‘net’ N retranslocation. In the present study, the amount and direction of gross N retranslocation within a canopy of deciduous oak Quercus serrata Thunb. ex. Murray saplings were investigated. Labeling was conducted with leaves of Q. serrata saplings cultivated under conditions of low-N (LN) or high-N (HN) fertility. Subsequently, N movement within the canopy was traced. Leaves at two different positions in the canopy (top and lateral) were labeled to determine the direction of gross N retranslocation. To detect seasonal differences, the leaf-labeling experiment was conducted twice during the early and late phases of the growing season. In addition, to compare the quantitative importance of gross N retranslocation and root N uptake, the latter was determined by labeling Q. serrata roots. The N-labeling experiment revealed gross N retranslocation among leaves, i.e., from top to lateral, lateral to top and lateral to lateral positions. Gross N retranslocation was quantitatively more important than root uptake, especially for plants cultivated at LN fertility. Season also affected the amount of gross N retranslocation, and these effects differed between LN and HN fertilities. These findings suggest that N allocation within a canopy is controlled dynamically by both gross N output and input. The mechanisms controlling gross N output and input likely function as key determinants of N allocation within a tree canopy.

Description: Natural temperature gradient (NTG) can be a significant problem in thermal sap flow measurements, particularly in dry environments with sparse vegetation. To resolve this problem, we propose a novel correction method called cyclic heat dissipation (CHD) in its thermal dissipation probe (TDP) application. The CHD method is based on cyclic, switching ON/OFF power schema measurements and a three-exponential model, extrapolating measured signal to steady state thermal equilibrium. The extrapolated signal OFF represents NTG, whereas the extrapolated signal ON represents standard TDP signal, biased by NTG. Therefore, subtraction of the OFF signal from the ON signal allows defining the unbiased TDP signal, finally processed according to standard Granier calibration. The in vivo Kalahari measurements were carried out in three steps on four different tree species, first as NTG, then as standard TDP and finally in CHD mode, each step for ~1–2 days. Afterwards, each tree was separated from its stem following modified Roberts' (1977) procedure, and CHD verification was applied. The typical NTG varying from ~0.5 °C during night-time to –1 °C during day-time, after CHD correction, resulted in significant reduction of sap flux densities ( J p ) as compared with the standard TDP, particularly distinct for low J p . The verification of the CHD method indicated ~20% agreement with the reference method, largely dependent on the sapwood area estimate. The proposed CHD method offers the following advantages: (i) in contrast to any other NTG correction method, it removes NTG bias from the measured signal by using in situ, extrapolated to thermal equilibrium signal; (ii) it does not need any specific calibration making use of the standard Granier calibration; (iii) it provides a physical background to the proposed NTG correction; (iv) it allows for power savings; (v) it is not tied to TDP, and so can be adapted to other thermal methods. In its current state, the CHD data processing is not yet fully automated.

Description: Patchy stomatal closure occurs in plants with heterobaric leaves, in which vertical extensions of bundle sheath cells delimit the mesophyll and restrict the diffusion of CO 2 . The scale of patchy stomatal behavior was investigated in this study. The distribution of PSII quantum yield ( II ) obtained from chlorophyll fluorescence images was used to evaluate the scale of stomatal patchiness and its relationship with leaf photosynthesis in the sun leaves of 2-year-old saplings of Quercus crispula Blume. Fluorescent patches were observed only during the day with low stomatal conductance. Comparison of numerical simulation of leaf gas exchange and chlorophyll fluorescence images showed that heterogeneous distribution of electron transport rate through PSII ( J ) was observed following stomatal closure with a bimodal manner under both natural and saturated photosynthetic photon flux densities. Thus, fluorescence patterns can be interpreted in terms of patchy stomatal closure. The mapping of J from chlorophyll fluorescence images showed that the scale of stomatal patchiness was approximately 2.5-fold larger than that of anatomical patches (lamina areas bounded by bundle sheath extensions within lamina). Our results suggest the spatial scale of stomatal patches in Q. crispula leaves.

Description: Predicted future changes in air temperature and atmospheric CO 2 concentration ([CO 2 ]), coupled with altered precipitation, are expected to substantially affect tree growth. Effects on growth may vary considerably across a species range, as temperatures vary from sub-optimal to supra-optimal for growth. We performed an experiment simultaneously at two locations in the current range of loblolly pine, a cool site and a warm site, to examine the effect of future climate conditions on growth of loblolly pine seedlings in contrasting regions of the species range. At both sites 1-year-old loblolly pine seedlings were grown in current (local ambient temperature and [CO 2 ]) and predicted future atmospheric conditions (ambient +2 °C temperature and 700 mol mol –1 [CO 2 ]). Additionally, high and low soil moisture treatments were applied within each atmospheric treatment at each site by altering the amount of water provided to the seedlings. Averaged across water treatments, photosynthesis ( A net ) was 31% greater at the cool site and 34% greater at the warm site in elevated temperature and [CO 2 ] compared with ambient temperature. Biomass accumulation was also stimulated by 38% at the cool site and by 24% at the warm site in that treatment. These results suggest that a temperature increase of 2 °C coupled with an increase in [CO 2 ] (predicted future climate) will create conditions favorable for growth of this species. Reduced soil moisture decreased growth in both current and predicted atmospheric conditions. Biomass accumulation and A net were reduced by ~39 and 17%, respectively, in the low water treatment. These results suggest that any benefit of future atmospheric conditions may be negated if soil moisture is reduced by altered precipitation patterns.

Description: Conifer bark beetles are often associated with fungal complexes whose components have different ecological roles. Some associated species are nutritionally obligate fungi, serving as nourishment to the larvae, whereas others are pathogenic blue-stain fungi known to be involved in the interaction with host defenses. In this study we characterized the local and systemic defense responses of Scots pine ( Pinus sylvestris L.) against Ophiostoma brunneo-ciliatum Math. (a blue-stain pathogen) and Hyalorhinocladiella macrospora (Franke-Grosm.) Harr. (a nutritional fungus). These fungi are the principal associates of the pine engraver beetle, Ips acuminatus (Gyll.). Host responses were studied following inoculation with the fungi, singly and as a fungal complex, and by identifying and quantifying terpenoids, phenolic compounds and lignin. Although the length of the necrotic lesions differed between control (wound) and fungal treatments, only two compounds (pinosylvin monomethyl ether and (+)-α-pinene) were significantly affected by the presence of the fungi, indicating that Scots pine has a generic, rather than specific, induced response. The fact that both nutritional and blue-stain fungi triggered comparable induced defense responses suggests that even a non-pathogenic fungus may participate in exhausting host plant defenses, indirectly assisting in the beetle establishment process. Our findings contribute to the further development of current theory on the role of associated fungal complexes in bark beetle ecology.

Description: Hydraulic traits were studied for six Nothofagus species from South America (Argentina and Chile), and for three of these species two populations were studied. The main goal was to determine if properties of the water conductive pathway in stems and leaves are functionally coordinated and to assess if leaves are more vulnerable to cavitation than stems, consistent with the theory of hydraulic segmentation along the vascular system of trees in ecosystems subject to seasonal drought. Vulnerability to cavitation, hydraulic conductivity of stems and leaves, leaf water potential, wood density and leaf water relations were examined. Large variations in vulnerability to cavitation of stems and leaves were observed across populations and species, but leaves were consistently more vulnerable than stems. Water potential at 50% loss of maximum hydraulic efficiency ( P 50 ) ranged from –0.94 to –2.44 MPa in leaves and from –2.6 to –5.3 MPa in stems across species and populations. Populations in the driest sites had sapwood and leaves more vulnerable to cavitation than those grown in the wettest sites. Stronger diurnal down-regulation in leaf hydraulic conductance compared with stem hydraulic conductivity apparently has the function to slow down potential water loss in stems and protect stem hydraulics from cavitation. Species-specific differences in wood density and leaf hydraulic conductance ( K Leaf ) were observed. Both traits were functionally related: species with higher wood density had lower K Leaf . Other stem and leaf hydraulic traits were functionally coordinated, resulting in Nothofagus species with an efficient delivery of water to the leaves. The integrity of the more expensive woody portion of the water transport pathway can thus be maintained at the expense of the replaceable portion (leaves) of the stem–leaf continuum under prolonged drought. Compensatory adjustments between hydraulic traits may help to decrease the rate of embolism formation in the trees more vulnerable to cavitation.

Description: Sap flow measurements conducted with thermal dissipation probes (TDPs) are vulnerable to natural temperature gradient (NTG) bias. Few studies, however, attempted to explain the dynamics underlying the NTG formation and its influence on the sensors' signal. This study focused on understanding how the TDP signals are affected by negative and positive temperature influences from NTG and tested the novel cyclic heat dissipation (CHD) method to filter out the NTG bias. A series of three experiments were performed in which gravity-driven water flow was enforced on freshly cut stem segments of Fagus sylvatica L., while an artificial temperature gradient (ATG) was induced. The first experiment sought to confirm the incidence of the ATG on sensors. The second experiment established the mis-estimations caused by the biasing effect of the ATG on standard TDP measurements. The third experiment tested the accuracy of the CHD method to account for the ATG biasing effect, as compared with other cyclic correction methods. During experiments, sap flow measured by TDP was assessed against gravimetric measurements. The results show that negative and positive ATGs were comparable in pattern but substantially larger than field NTGs. Second, the ATG bias caused an overestimation of the standard TDP sap flux density of ~17 cm 3 cm –2 h –1 by 76%, and the sap flux density of ~2 cm 3 cm –2 h –1 by over 800%. Finally, the proposed CHD method successfully reduced the max. ATG bias to 25% at ~11 cm 3 cm –2 h –1 and to 40% at ~1 cm 3 cm –2 h –1 . We concluded that: (i) the TDP method is susceptible to NTG especially at low flows; (ii) the CHD method successfully corrected the TDP signal and resulted in generally more accurate sap flux density estimates (mean absolute percentage error ranging between 11 and 21%) than standard constant power TDP method and other cyclic power methods; and (iii) the ATG enforcing system is a suitable way of re-creating NTG for future tests.

Description: Two mature clones of Norway spruce ( Picea abies (L.) Karst.) that have previously been shown to have differential degrees of resistance towards the necrotrophic pathogen Heterobasidion parviporum (Niemelä & Korhonen) were compared with respect to the primed defense expression of transcripts related to biosynthesis of lignin, stilbenes and other phenolic compounds from one year to the next. The host's response to physical wounding and pathogen inoculation was examined in the initial year, whereas indications of heightened basal defense level or primed response, and responses to re-wounding, were examined the following year. The responses of the two clones to wounding and pathogen inoculation, examined in the initial year, differed; the increases in lignin and phenolics were more distinct in response to the pathogen than to wounding alone. The more resistant clone 589 had higher initial lignin concentrations in the cell walls when compared with clone 409, and these remained higher in clone 589 over both years and increased after the treatments. Both clones responded at the transcriptional and chemical levels to wounding; changes were evident both in the initial wounds and when re-wounded the following year. There were distinct differences in the basal transcript levels of the lignin pathway-related genes, phenolics and total lignin levels in healthy tissue from the initial year to the following year indicative of a primed host response or at least altered constitutive level of defense expression.

Description: In a world of diminishing water reservoirs and a rising demand for food, the practice and development of water stress indicators and sensors are in rapid progress. The heat dissipation method, originally established by Granier, is herein applied and modified to enable sap flow measurements in date palm trees in the southern Arava desert of Israel. A long and tough sensor was constructed to withstand insertion into the date palm's hard exterior stem. This stem is wide and fibrous, surrounded by an even tougher external non-conducting layer of dead leaf bases. Furthermore, being a monocot species, water flow does not necessarily occur through the outer part of the palm's stem, as in most trees. Therefore, it is highly important to investigate the variations of the sap flux densities and determine the preferable location for sap flow sensing within the stem. Once installed into fully grown date palm trees stationed on weighing lysimeters, sap flow as measured by the modified sensors was compared with the actual transpiration. Sap flow was found to be well correlated with transpiration, especially when using a recent calibration equation rather than the original Granier equation. Furthermore, inducing the axial variability of the sap flux densities was found to be highly important for accurate assessments of transpiration by sap flow measurements. The sensors indicated no transpiration at night, a high increase of transpiration from 06:00 to 09:00, maximum transpiration at 12:00, followed by a moderate reduction until 08:00; when transpiration ceased. These results were reinforced by the lysimeters' output. Reduced sap flux densities were detected at the stem's mantle when compared with its center. These results were reinforced by mechanistic measurements of the stem's specific hydraulic conductivity. Variance on the vertical axis was also observed, indicating an accelerated flow towards the upper parts of the tree and raising a hypothesis concerning dehydrating mechanisms of the date palm tree. Finally, the sensors indicated reduction in flow almost immediately after irrigation of field-grown trees was withheld, at a time when no climatic or phenological conditions could have led to reduction in transpiration.

Description: Northern forests are currently experiencing increasing mean temperatures, especially during autumn and spring. Consequently, alterations in carbon sequestration, leaf biochemical quality and freezing tolerance (FT) are likely to occur. The interactive effects of elevated temperature and ozone (O 3 ), the most harmful phytotoxic air pollutant, on Norway spruce ( Picea abies (L.) Karst.) seedlings were studied by analysing phenology, metabolite concentrations in the needles, FT and gas exchange. Sampling was performed in September and May. The seedlings were exposed to a year-round elevated temperature (+1.3 °C), and to 1.4 x ambient O 3 concentration during the growing season in the field. Elevated temperature increased the concentrations of amino acids, organic acids of the citric acid cycle and some carbohydrates, and reduced the concentrations of phenolic compounds, some organic acids of the shikimic acid pathway, sucrose, cyclitols and steroids, depending on the timing of the sampling. Although growth onset occurred earlier at elevated temperature, the temperature of 50% lethality (LT 50 ) was similar in the treatments. Photosynthesis and the ratio of photosynthesis to dark respiration were reduced by elevated temperature. Elevated concentrations of O 3 reduced the total concentration of soluble sugars, and tended to reduce LT 50 of the needles in September. These results show that alterations in needle chemical quality can be expected at elevated temperatures, but the seedlings' sensitivity to autumn and spring frosts is not altered. Elevated O 3 has the potential to disturb cold hardening of Norway spruce seedlings in autumn, and to alter the water balance of the seedling through changes in stomatal conductance ( g s ), while elevated temperature is likely to reduce g s and consequently reduce the O 3 -flux inside the leaves.

Description: Sunflecks are brief, intermittent periods of high photon flux density (PFD) that can significantly improve carbon gain in shaded forest understories and lower canopies of trees. In this review, we discuss the physiological basis of leaf-level responses to sunflecks and the mechanisms plants use to tolerate sudden changes in PFD and leaf temperature induced by sunflecks. We also examine the potential effects of climate change stresses (including elevated temperatures, rising CO 2 concentrations and drought) on the ability of tree species to use sunflecks, and advocate more research to improve our predictions of seedling and tree carbon gain in future climates. Lastly, while we have the ability to model realistic responses of photosynthesis to fluctuating PFD, dynamic responses of photosynthesis to sunflecks are not accounted for in current models of canopy carbon uptake, which can lead to substantial overestimates of forest carbon fixation. Since sunflecks are a critical component of seasonal carbon gain for shaded leaves, sunfleck regimes and physiological responses to sunflecks should be incorporated into models to more accurately capture forest carbon dynamics.

Description: In this study we investigated the role that ascorbate (AA) and glutathione (GSH) play in the plant pathogen interaction of susceptible Eucalyptus sieberi L. A. Johnson and resistant Eucalyptus sideroxylon Woolls with Phytophthora cinnamomi Rands root infection. In a glasshouse study, seedlings were grown in soil-free plant boxes to facilitate the inoculation of the root systems by a P. cinnamomi zoospore solution. Ascorbate and GSH concentrations were measured in infected roots and leaves, along with leaf gas exchange, chlorophyll fluorescence and carbohydrate concentrations over a time course up to 312 h (13 days) post-inoculation (pi). At the early stages of infection (from 24 h pi), significant decreases in AA and GSH concentrations were observed in the infected roots and leaves of the susceptible E. sieberi seedlings. At the later stage of infection (312 h pi), the earlier AA decreases in the leaves of infected plants had become significant increases. In contrast, late, significant AA increases in the absence of any GSH changes were observed in the infected roots of the resistant E. sideroxylon seedlings. In E. sideroxylon leaves, a significant GSH increase occurred at 24 h pi; however, by 312 h pi the earlier increase had become a significant decrease, while no changes occurred in AA. In E. sieberi , photosynthesis ( A ), stomatal conductance ( g s ) and PSII quantum efficiency ( PSII ) were reduced by ~60, 80 and 30%, respectively, in infected plants and remained significantly lower than uninfected controls for the duration of the experiment. Significant reductions in these parameters did not occur until later (120 h pi for g s and 312 h pi for A and PSII ), and to a lesser extent in the resistant species. Non-structural carbohydrate analysis of roots and leaves indicate that carbohydrate metabolism and resource flow between shoots and roots may have been altered at later infection stages. This study suggests that reduced antioxidant capacity, leaf physiological function and carbohydrate metabolism are associated with susceptibility in E. sieberi to P. cinnamomi infection, while AA increases and new root formation were associated with resistance in E . sideroxylon .

Description: Interspecific gene flow is common in oaks. In the Mediterranean, this process produced geographical differentiations and new species, which may have contributed to the diversification of the production of volatile terpenes in the oak species of this region. The endemic North African deciduous oak Quercus afares (Pomel) is considered to be a stabilized hybrid between the evergreen Quercus suber (L.) and the deciduous Quercus canariensis (Willd.), presumably being monoterpene and isoprene emitters, respectively. In a common garden experiment, we examined the terpene emission capacities, terpene synthase (TPS) activities and nuclear genetic markers in 52 trees of these three oak species. All but one of the Q. suber and Q. canariensis trees were found to be genetically pure, whereas most Q. afares trees possessed a mixed genotype with a predominance of Q. suber alleles. Analysis of the foliar terpene emissions and TPS activities revealed that all the Q. canariensis trees strongly produced isoprene while all the Q. suber trees were strong monoterpene producers. Quercus afares trees produced monoterpenes as well but at more variable and significantly lower rates, and with a monoterpene pattern different than that observed in Q. suber . Among 17 individuals tested, one Q. afares tree emitted only an insignificant amount of terpenes. No mixed isoprene/monoterpene emitter was detected. Our results suggest that the capacity and pattern of volatile terpene production in Algerian Q. afares populations have strongly diverged from those of its parental species and became quantitatively and qualitatively reduced, including the complete suppression of isoprene production.

Description: Low temperature plays a crucial role in seasonal development of woody plants and may directly impact crop production, more particularly in temperate fruit trees. Given its high genetic variability and adaptability to different climatic conditions, almond ( Prunus dulcis Mill.) is an interesting model to understand the mechanisms regulating low temperature sensing in fruit trees. In this paper, we report the cloning and characterization of two genes ( PdCBF1 and PdCBF2 ) belonging to the C-repeat-binding factor (CBF) family of transcription factors. Southern blotting analysis showed that this family is composed of at least five members. In almond shoots propagated in vitro, transcription of these genes was rapidly induced by low temperature, suggesting an involvement in cold acclimation. Transactivation assays showed that PdCBF1 and PdCBF2 could bind to dehydration responsive element/C-repeat containing sequences, as activators of gene expression. In addition, induction of both PdCBF s by cold was higher towards the end of the day, which agreed with the expression pattern of PdDehydrin1 , a predicted CBF target gene. Furthermore, PdCBF1 and PdCBF2 were also transiently induced by abscisic acid and drought treatments. Considering the bin mapping analysis that correlated PdCBF s and PdDHN1 (respectively in linkage groups 5 and 7) with two different quantitative trait locicontrolling blooming time, it is relevant to perform further association studies that may validate their effect on this trait.

Description: Leaves, the distal section of the soil–plant–atmosphere continuum, exhibit the lowest water potentials in a plant. In contrast to angiosperm leaves, knowledge of the hydraulic architecture of conifer needles is scant. We investigated the hydraulic efficiency and safety of Pinus pinaster needles, comparing different techniques. The xylem hydraulic conductivity ( k s ) and embolism vulnerability ( P 50 ) of both needle and stem were measured using the cavitron technique. The conductance and vulnerability of whole needles were measured via rehydration kinetics, and Cryo-SEM and 3D X-ray microtomographic observations were used as reference tools to validate physical measurements. The needle xylem of P. pinaster had lower hydraulic efficiency ( k s = 2.0 x 10 –4  m 2  MPa –1  s –1 ) and safety ( P 50 = – 1.5 MPa) than stem xylem ( k s = 7.7 x 10 –4  m 2  MPa –1  s –1 ; P 50 = – 3.6 to – 3.2 MPa). P 50 of whole needles (both extra-vascular and vascular pathways) was – 0.5 MPa, suggesting that non-vascular tissues were more vulnerable than the xylem. During dehydration to – 3.5 MPa, collapse and embolism in xylem tracheids, and gap formation in surrounding tissues were observed. However, a discrepancy in hydraulic and acoustic results appeared compared with visualizations, arguing for greater caution with these techniques when applied to needles. Our results indicate that the most distal parts of the water transport pathway are limiting for hydraulics of P. pinaster . Needle tissues exhibit a low hydraulic efficiency and low hydraulic safety, but may also act to buffer short-term water deficits, thus preventing xylem embolism.

Description: The lignification process in poplar tension wood lignified cell wall layers, specifically the S 1 and S 2 layers and the compound middle lamella (CML), was analysed using ultraviolet (UV) and transmission electron microscopy (TEM). Variations in the thickness of the gelatinous layer (G-layer) were also measured to clarify whether the lignified cell wall layers had completed their lignification before the deposition of G-layers, or, on the contrary, if lignification of these layers was still active during G-layer formation. Observations using UV microscopy and TEM indicated that both UV absorbance and the degree of potassium permanganate staining increased in the CML and S 1 and S 2 layers during G-layer formation, suggesting that the lignification of these lignified layers is still in progress during G-layer formation. In the context of the cell-autonomous monolignol synthesis hypothesis, our observations suggest that monolignols must go through the developing G-layer during the lignification of CML and the S 1 and S 2 layers. The alternative hypothesis of external synthesis (in the rays) does not require that monolignols go through the G-layer before being deposited in the CML, or the S 1 and S 2 layers. Interestingly, the previous observation of lignin in the poplar G-layer was not confirmed with the microscopy techniques used in the present study.

Description: Oak forests dominate much of the eastern USA, but their future is uncertain due to a number of threats and widespread failure of oak regeneration. A sudden loss of oaks ( Quercus spp . ) could be accompanied by major changes in forest nitrogen (N) cycles with important implications for plant nutrient uptake and tree species composition. In this study, we measured the changes in N use and growth rates of black birch trees ( Betula lenta L.) following oak girdling at the Black Rock Forest in southeastern New York, USA. Data were collected from nine experimental plots composed of three treatments: 100% oaks girdled (OG), 50% oaks girdled (O50) and control (C). Foliar N concentration and foliar 15 N abundance increased significantly in the oak-girdled plots relative to the control, indicating that the loss of oaks significantly altered N cycling dynamics. As mineralization and nitrification rates increase following oak loss, black birch trees increase N absorption as indicated by higher foliar N content and increased growth rates. Foliar N concentration increased by 15.5% in the O50 and 30.6% in the OG plots relative to the control, while O50 and OG plots were enriched in 15 N by 1.08 and 3.33, respectively ( P 〈 0.0001). A 641% increase in black birch growth rates in OG plots suggests that this species is able to respond to additional N availability and/or increased light availability. The loss of oaks and subsequent increase in black birch productivity may have a lasting impact on ecosystem form and function.

Description: It is important to identify the mechanism by which ionising irradiation induces various genomic alterations in the progeny of surviving cells. Ionising irradiation activates mobile elements like retrotransposons, although the mechanism of its phenomena consisting of transcriptions and insertions of the products into new sites of the genome remains unclear. In this study, we analysed the effects of sparsely ionising X-rays and densely ionising carbon-ion beams on the activities of a family of active retrotransposons, long interspersed nuclear elements 1 (L1). We used the L1/reporter knock-in human glioma cell line, NP-2/L1RP-enhanced GFP (EGFP), that harbours full-length L1 tagged with EGFP retrotransposition detection cassette (L1RP-EGFP) in the chromosomal DNA. X-rays and carbon-ion beams similarly increased frequencies the transcription from L1RP-EGFP and its retrotransposition. Short-sized de novo L1RP-EGFP insertions with 5'-truncation were induced by X-rays, while full-length or long-sized insertions (〉5 kb, containing ORF1 and ORF2) were found only in cell clones irradiated by the carbon-ion beams. These data suggest that X-rays and carbon-ion beams induce different length of de novo L1 insertions, respectively. Our findings thus highlight the necessity to investigate the mechanisms of mutations caused by transposable elements by ionising irradiation.

Description: The ultraviolet (UV)-B spectrum in solar UV radiation is essential for stimulating the epidermal production of vitamin D but also damages DNA and causes cancer in exposed cells. We examined the role of solar UV in inducing DNA damage in blood lymphocytes and the possible modulation of this damage by serum 25-hydroxy vitamin D (25(OH)D) in 207 male and female participants from South Australia. Personal solar UV exposure was estimated from hours of outdoor exposure recalled at the time of blood collection for analysis of DNA damage in lymphocytes, using the cytokinesis-block micronucleus cytome (CBMN-cyt) assay and of serum 25(OH)D. We examined the association between solar UV exposure, serum 25(OH)D and DNA damage using multiple linear regression, with age, sex, body mass index and alcohol consumption as covariates. The frequency of cells with micronuclei (a biomarker of chromosome breakage or loss) increased with increasing sun exposure [% increase = 5.24; 95% confidence interval (CI): 0.35 to 10.37 P -value = 0.04] but cells with nucleoplasmic bridges (a biomarker of misrepair of DNA strand breaks or telomere end fusions) decreased (% increase = –8.38; 95% CI: –14.32 to –2.03 P -value = 0.01). There was also a fall in the nuclear division index (NDI) (% increase = –1.01; 95% CI: –2.00 to 0.00 P -value = 0.05), suggesting diminished mitogenic response and, possibly, immune suppression. There was no overall relationship between 25(OH)D and DNA damage. There were, however, weak modulating effects of 25(OH)D on the associations of solar UV exposure with micronucleus formation and with NDI ( P -interaction = 0.03 and 0.05, respectively), where the increase in micronuclei and fall in NDI with increasing solar UV were greater at serum 25(OH)D levels 〈50 nmol/l. Thus, the influence of solar UV exposure in causing DNA damage or immune suppression in internal tissues may be stronger when vitamin D levels are low.

Description: We have developed and validated a sandwich chemiluminescence immunoassay (SCIA) which measures polycyclic aromatic hydrocarbon (PAH)–DNA adducts combining high throughput and adequate sensitivity, appropriate for evaluation of adduct levels in human population studies. Fragmented DNA is incubated with rabbit antiserum elicited against DNA modified with r7,t8-dihydroxy-t-9,10-epoxy-7,8,9,10-tetrahydrobenzo[ a ]pyrene (BPDE) and subsequently trapped by goat anti-rabbit IgG bound to a solid surface. Anti-single-stranded (ss) DNA antibodies binds in a quantity proportional to the adduct levels and is detected by chemiluminescence. The BPDE–DNA SCIA has a limit of detection of 3 adducts per 10 9 nucleotides with 5 μg DNA per well. We have validated the BPDE–DNA SCIA using DNA modified in vitro , DNA from benzo[ a ]pyrene (BP)-exposed cultured cells and mice. The levels of adduct measured by SCIA were lower (30–60%) than levels of bulky DNA adducts measured in the same samples by 32 P-postlabelling. The BPDE–DNA SCIA also detected adducts produced in vivo by PAHs other than BP. When blood DNA samples from maternal/infant pairs were assayed by BPDE–DNA SCIA, the adduct levels obtained were significantly correlated. However, there was no correlation between 32 P-postlabelling and SCIA values for the same samples. The SCIA can be extended to any DNA adduct and is expected to provide, when fully automated, a valuable high-throughput approach in large-scale population studies.

Description: Introducing nitrogen-fixing tree species in fast-growing eucalypt plantations has the potential to improve soil nitrogen availability compared with eucalypt monocultures. Whether or not the changes in soil nutrient status and stand structure will lead to mixtures that out-yield monocultures depends on the balance between positive interactions and the negative effects of interspecific competition, and on their effect on carbon (C) uptake and partitioning. We used a C budget approach to quantify growth, C uptake and C partitioning in monocultures of Eucalyptus grandis (W. Hill ex Maiden) and Acacia mangium (Willd.) (treatments E100 and A100, respectively), and in a mixture at the same stocking density with the two species at a proportion of 1 : 1 (treatment MS). Allometric relationships established over the whole rotation, and measurements of soil CO 2 efflux and aboveground litterfall for ages 4–6 years after planting were used to estimate aboveground net primary production (ANPP), total belowground carbon flux (TBCF) and gross primary production (GPP). We tested the hypotheses that (i) species differences for wood production between E. grandis and A. mangium monocultures were partly explained by different C partitioning strategies, and (ii) the observed lower wood production in the mixture compared with eucalypt monoculture was mostly explained by a lower partitioning aboveground. At the end of the rotation, total aboveground biomass was lowest in A100 (10.5 kg DM m –2 ), intermediate in MS (12.2 kg DM m –2 ) and highest in E100 (13.9 kg DM m –2 ). The results did not support our first hypothesis of contrasting C partitioning strategies between E. grandis and A. mangium monocultures: the 21% lower growth ( B w ) in A100 compared with E100 was almost entirely explained by a 23% lower GPP, with little or no species difference in ratios such as TBCF/GPP, ANPP/TBCF, B w /ANPP and B w /GPP. In contrast, the 28% lower B w in MS than in E100 was explained both by a 15% lower GPP and by a 15% lower fraction of GPP allocated to wood growth, thus partially supporting our second hypothesis: mixing the two species led to shifts in C allocations from above- to belowground, and from growth to litter production, for both species.

Description: Soil temperature is proposed to affect the photosynthetic rate and carbon allocation in boreal trees through sink limitation. The aim of this study was to investigate the effect of temperature on CO 2 exchange, biomass partitioning and ectomycorrhizal (ECM) fungi of boreal tree species. We measured carbon allocation, above- and below-ground CO 2 exchange and the species composition of associated ECM fungi in the rhizosphere of Scots pine ( Pinus sylvestris L.), Norway spruce ( Picea abies K.) and silver birch ( Betula pendula Roth) seedlings grown in soil maintained at 7–12, 12–15 and 16–22 °C. We found increased root biomass and photosynthetic rate at higher soil temperatures, but simultaneously with photosynthesis rate, higher temperature generally increased soil respiration as well as shoot, and root and rhizosphere respiration. The net CO 2 exchange and seedling biomass did not increase significantly with increasing temperature due to a concomitant increase in carbon assimilation and respiration rates. The 2-month-long growth period in different soil temperatures did not alter the ECM fungi species composition and the below-ground carbon sink strength did not seem to be directly related to ECM biomass and species composition in any of the tree species. Ectomycorrhizal species composition and number of mycorrhiza did not explain the CO 2 exchange results at different temperatures.

Description: Wood production represents a large but variable fraction of gross primary production (GPP) in highly productive Eucalyptus plantations. Assessing patterns of carbon (C) partitioning (C flux as a fraction of GPP) between above- and belowground components is essential to understand mechanisms driving the C budget of these plantations. Better knowledge of fluxes and partitioning to woody and non-woody tissues in response to site characteristics and resource availability could provide opportunities to increase forest productivity. Our study aimed at investigating how C allocation varied within one apparently homogeneous 90 ha stand of Eucalyptus grandis (W. Hill ex Maiden) in Southeastern Brazil. We assessed annual above­ground net primary production (ANPP: stem, leaf, and branch production) and total belowground C flux (TBCF: the sum of root production and respiration and mycorrhizal production and respiration), GPP (computed as the sum of ANPP, TBCF and estimated aboveground respiration) on 12 plots representing the gradient of productivity found within the stand. The spatial heterogeneity of topography and associated soil attributes across the stand likely explained this fertility gradient. Component fluxes of GPP and C partitioning were found to vary among plots. Stem NPP ranged from 554 g C m –2 year –1 on the plot with lowest GPP to 923 g C m –2 year –1 on the plot with highest GPP. Total belowground carbon flux ranged from 497 to 1235 g C m –2 year –1 and showed no relationship with ANPP or GPP. Carbon partitioning to stem NPP increased from 0.19 to 0.23, showing a positive trend of increase with GPP ( R 2 = 0.29, P = 0.07). Variations in stem wood production across the gradient of productivity observed at our experimental site were a result of the variability in C partitioning to different forest system components.

Description: We review approaches to predicting carbon and nitrogen allocation in forest models in terms of their underlying assumptions and their resulting strengths and limitations. Empirical and allometric methods are easily developed and computationally efficient, but lack the power of evolution-based approaches to explain and predict multifaceted effects of environmental variability and climate change. In evolution-based methods, allocation is usually determined by maximization of a fitness proxy, either in a fixed environment, which we call optimal response (OR) models, or including the feedback of an individual's strategy on its environment (game-theoretical optimization, GTO). Optimal response models can predict allocation in single trees and stands when there is significant competition only for one resource. Game-theoretical optimization can be used to account for additional dimensions of competition, e.g., when strong root competition boosts root allocation at the expense of wood production. However, we demonstrate that an OR model predicts similar allocation to a GTO model under the root-competitive conditions reported in free-air carbon dioxide enrichment (FACE) experiments. The most evolutionarily realistic approach is adaptive dynamics (AD) where the allocation strategy arises from eco-evolutionary dynamics of populations instead of a fitness proxy. We also discuss emerging entropy-based approaches that offer an alternative thermodynamic perspective on allocation, in which fitness proxies are replaced by entropy or entropy production. To help develop allocation models further, the value of wide-ranging datasets, such as FLUXNET, could be greatly enhanced by ancillary measurements of driving variables, such as water and soil nitrogen availability.

Description: Deep root water uptake and hydraulic redistribution (HR) have been shown to play a major role in forest ecosystems during drought, but little is known about the impact of climate change, fertilization and soil characteristics on HR and its consequences on water and carbon fluxes. Using data from three mid-rotation loblolly pine plantations, and simulations with the process-based model MuSICA, this study indicated that HR can mitigate the effects of soil drying and had important implications for carbon uptake potential and net ecosystem exchange (NEE), especially when N fertilization is considered. At the coastal site (C), characterized by deep organic soil, HR increased dry season tree transpiration ( T ) by up to 40%, and such an increase affected NEE through major changes in gross primary productivity (GPP). Deep-rooted trees did not necessarily translate into a large volume of HR unless soil texture allowed large water potential gradients to occur, as was the case at the sandy site (S). At the Piedmont site (P) characterized by a shallow clay-loam soil, HR was low but not negligible, representing up to 10% of T . In the absence of HR, it was predicted that at the C, S and P sites, annual GPP would have been diminished by 19, 7 and 9%, respectively. Under future climate conditions HR was predicted to be reduced by up to 25% at the C site, reducing the resilience of trees to precipitation deficits. The effect of HR on T and GPP was predicted to diminish under future conditions by 12 and 6% at the C and P sites, respectively. Under future conditions, T was predicted to stay the same at the P site, but to be marginally reduced at the C site and slightly increased at the S site. Future conditions and N fertilization would decrease T by 25% at the C site, by 15% at the P site and by 8% at the S site. At the C and S sites, GPP was estimated to increase by 18% and by 〉70% under future conditions, respectively, with little effect of N fertilization. At the P site, future conditions would stimulate GPP by only 12%, but future conditions plus N fertilization would increase GPP by 24%. As a consequence, in all sites, water use efficiency was predicted to improve dramatically with future conditions. Modeling the effect of reduced annual precipitation indicated that limited water availability would decrease all carbon fluxes, including NEE and respiration. Our simulations highlight the interactive effects of nutrients and elevated CO 2 , and showed that the effect of N fertilization would be greater under future climate conditions.

Description: Warmer climates induced by elevated atmospheric CO 2 (eCO 2 ) are expected to increase damaging bark beetle activity in pine forests, yet the effect of eCO 2 on resin production—the tree's primary defense against beetle attack—remains largely unknown. Following growth-differentiation balance theory, if extra carbohydrates produced under eCO 2 are not consumed by respiration or growth, resin production could increase. Here, the effect of eCO 2 on resin production of mature pines is assessed. As predicted, eCO 2 enhanced resin flow by an average of 140% ( P = 0.03) in canopy dominants growing in low-nitrogen soils, but did not affect resin flow in faster-growing fertilized canopy dominants or in carbohydrate-limited suppressed individuals. Thus, pine trees may become increasingly protected from bark beetle attacks in an eCO 2 climate, except where they are fertilized or are allowed to become overcrowded.

Description: Research on the degree to which carbon (C) availability limits growth in trees, as well as recent trends in climate change and concurrent increases in drought-related tree mortality, have led to a renewed focus on the physiological mechanisms associated with tree growth responses to current and future climate. This has led to some dispute over the role of stored non-structural C compounds as indicators of a tree's current demands for photosynthate. Much of the uncertainty surrounding this issue could be resolved by developing a better understanding of the potential functions of non-structural C stored within trees. In addition to functioning as a buffer to reconcile temporal asynchrony between C demand and supply, the storage of non-structural C compounds may be under greater regulation than commonly recognized. We propose that in the face of environmental stochasticity, large, long-lived trees may require larger C investments in storage pools as safety margins than previously recognized, and that an important function of these pools may be to maintain hydraulic transport, particularly during episodes of severe stress. If so, survival and long-term growth in trees remain a function of C availability. Given that drought, freeze–thaw events and increasing tree height all impose additional constraints on vascular transport, the common trend of an increase in non-structural carbohydrate concentrations with tree size, drought or cold is consistent with our hypothesis. If the regulated maintenance of relatively large constitutive stored C pools in trees serves to maintain hydraulic integrity, then the minimum thresholds are expected to vary depending on the specific tissues, species, environment, growth form and habit. Much research is needed to elucidate the extent to which allocation of C to storage in trees is a passive vs. an active process, the specific functions of stored C pools, and the factors that drive active C allocation to storage.

Description: In the present experiment, the single and combined effects of elevated temperature and ozone (O 3 ) on four silver birch genotypes (gt12, gt14, gt15 and gt25) were studied in an open-air field exposure design. Above- and below-ground biomass accumulation, stem growth and soil respiration were measured in 2008. In addition, a 13 C-labelling experiment was conducted with gt15 trees. After the second exposure season, elevated temperature increased silver birch above- and below-ground growth and soil respiration rates. However, some of these variables showed that the temperature effect was modified by tree genotype and prevailing O 3 level. For instance, in gt14 soil respiration was increased in elevated temperature alone (T) and in elevated O 3 and elevated temperature in combination (O 3 + T) treatments, but in other genotypes O 3 either partly (gt12) or totally nullified (gt25) temperature effects on soil respiration, or acted synergistically with temperature (gt15). Before leaf abscission, all genotypes had the largest leaf biomass in T and O 3 + T treatments, whereas at the end of the season temperature effects on leaf biomass depended on the prevailing O 3 level. Temperature increase thus delayed and O 3 accelerated leaf senescence, and in combination treatment O 3 reduced the temperature effect. Photosynthetic : non-photosynthetic tissue ratios (P : nP ratios) showed that elevated temperature increased foliage biomass relative to woody mass, particularly in gt14 and gt12, whereas O 3 and O 3 + T decreased it most clearly in gt25. O 3 -caused stem growth reductions were clearest in the fastest-growing gt14 and gt25, whereas mycorrhizal root growth and sporocarp production increased under O 3 in all genotypes. A labelling experiment showed that temperature increased tree total biomass and hence 13 C fixation in the foliage and roots and also label return was highest under elevated temperature. Ozone seemed to change tree 13 C allocation, as it decreased foliar 13 C excess amount, simultaneously increasing 13 C excess obtained from the soil. The present results suggest that warming has potential to increase silver birch growth and hence carbon (C) accumulation in tree biomass, but the final magnitude of this C sink strength is partly counteracted by temperature-induced increase in soil respiration rates and simultaneous O 3 stress. Silver birch populations' response to climate change will also largely depend on their genotype composition.

Description: The dynamics of rapid changes in carbon (C) partitioning within forest ecosystems are not well understood, which limits improvement of mechanistic models of C cycling. Our objective was to inform model processes by describing relationships between C partitioning and accessible environmental or physiological measurements, with a special emphasis on short-term C flux through a forest ecosystem. We exposed eight 7-year-old loblolly pine ( Pinus taeda L.) trees to air enriched with 13 CO 2 and then implemented adjacent light shade (LS) and heavy shade (HS) treatments in order to manipulate C uptake and flux. The impacts of shading on photosynthesis, plant water potential, sap flow, basal area growth, root growth and soil CO 2 efflux rate (CER) were assessed for each tree over a 3-week period. The progression of the 13 C label was concurrently tracked from the atmosphere through foliage, phloem, roots and surface soil CO 2 efflux. The HS treatment significantly reduced C uptake, sap flow, stem growth and fine root standing crop, and resulted in greater residual soil water content to 1 m depth. Soil CER was strongly correlated with sap flow on the previous day, but not the current day, with no apparent treatment effect on the relationship. Although there were apparent reductions in new C flux belowground, the HS treatment did not noticeably reduce the magnitude of belowground autotrophic and heterotrophic respiration based on surface soil CER, which was overwhelmingly driven by soil temperature and moisture. The 13 C label was immediately detected in foliage on label day (half-life = 0.5 day), progressed through phloem by Day 2 (half-life = 4.7 days), roots by Days 2–4, and subsequently was evident as respiratory release from soil which peaked between Days 3 and 6. The 13 C of soil CO 2 efflux was strongly correlated with phloem 13 C on the previous day, or 2 days earlier. While the 13 C label was readily tracked through the ecosystem, the fate of root C through respiratory, mycorrhizal or exudative release pathways was not assessed. These data detail the timing and relative magnitude of C flux through various components of a young pine stand in relation to environmental conditions.

Description: Understanding the underlying mechanisms that account for the impact of potassium (K) fertilization and its replacement by sodium (Na) on tree growth is key to improving the management of forest plantations that are expanding over weathered tropical soils with low amounts of exchangeable bases. A complete randomized block design was planted with Eucalyptus grandis (W. Hill ex Maiden) to quantify growth, carbon uptake and carbon partitioning using a carbon budget approach. A combination of approaches including the establishment of allometric relationships over the whole rotation and measurements of soil CO 2 efflux and aboveground litterfall at the end of the rotation were used to estimate aboveground net production (ANPP), total belowground carbon flux and gross primary production (GPP). The stable carbon isotope ( 13 C) of stem wood α-cellulose produced every year was used as a proxy for stomatal limitation of photosynthesis. Potassium fertilization increased GPP and decreased the fraction of carbon allocated belowground. Aboveground net production was strongly enhanced, and because leaf lifespan increased, leaf biomass was enhanced without any change in leaf production, and wood production (P W ) was dramatically increased. Sodium application decreased the fraction of carbon allocated belowground in a similar way, and enhanced GPP, ANPP and P W , but to a lesser extent compared with K fertilization. Neither K nor Na affected 13 C of stem wood α-cellulose, suggesting that water-use efficiency was the same among the treatments and that the inferred increase in leaf photosynthesis was not only related to a higher stomatal conductance. We concluded that the response to K fertilization and Na addition on P W resulted from drastic changes in carbon allocation.

Description: Pulse-labelling of trees with stable or radioactive carbon (C) isotopes offers the unique opportunity to trace the fate of labelled CO 2 into the tree and its release to the soil and the atmosphere. Thus, pulse-labelling enables the quantification of C partitioning in forests and the assessment of the role of partitioning in tree growth, resource acquisition and C sequestration. However, this is associated with challenges as regards the choice of a tracer, the methods of tracing labelled C in tree and soil compartments and the quantitative analysis of C dynamics. Based on data from 47 studies, the rate of transfer differs between broadleaved and coniferous species and decreases as temperature and soil water content decrease. Labelled C is rapidly transferred belowground—within a few days or less—and this transfer is slowed down by drought. Half-lives of labelled C in phloem sap (transfer pool) and in mature leaves (source organs) are short, while those of sink organs (growing tissues, seasonal storage) are longer. 13 C measurements in respiratory efflux at high temporal resolution provide the best estimate of the mean residence times of C in respiratory substrate pools, and the best basis for compartmental modelling. Seasonal C dynamics and allocation patterns indicate that sink strength variations are important drivers for C fluxes. We propose a conceptual model for temperate and boreal trees, which considers the use of recently assimilated C versus stored C. We recommend best practices for designing and analysing pulse-labelling experiments, and identify several topics which we consider of prime importance for future research on C allocation in trees: (i) whole-tree C source–sink relations, (ii) C allocation to secondary metabolism, (iii) responses to environmental change, (iv) effects of seasonality versus phenology in and across biomes, and (v) carbon–nitrogen interactions. Substantial progress is expected from emerging technologies, but the largest challenge remains to carry out in situ whole-tree labelling experiments on mature trees to improve our understanding of the environmental and physiological controls on C allocation.