Ancient events and climate adaptive capacity shaped distinct chloroplast genetic structure in the oak lineages.

BACKGROUND: Understanding the origin of genetic variation is the key to predict how species will respond to future climate change. The genus Quercus is a species-rich and ecologically diverse woody genus that dominates a wide range of forests and woodland communities of the Northern Hemisphere. Quercus thus offers a unique opportunity to investigate how adaptation to environmental changes has shaped the spatial genetic structure of closely related lineages. Furthermore, Quercus provides a deep insight into how tree species will respond to future climate change. This study investigated whether closely related Quercus lineages have similar spatial genetic structures and moreover, what roles have their geographic distribution, ecological tolerance, and historical environmental changes played in the similar or distinct genetic structures. RESULTS: Despite their close relationships, the three main oak lineages (Quercus sections Cyclobalanopsis, Ilex, and Quercus) have different spatial genetic patterns and occupy different climatic niches. The lowest level and most homogeneous pattern of genetic diversity was found in section Cyclobalanopsis, which is restricted to warm and humid climates. The highest genetic diversity and strongest geographic genetic structure were found in section Ilex, which is due to their long-term isolation and strong local adaptation. The widespread section Quercus is distributed across the most heterogeneous range of environments; however, it exhibited moderate haplotype diversity. This is likely due to regional extinction during Quaternary climatic fluctuation in Europe and North America. CONCLUSIONS: Genetic variations of sections Ilex and Quercus were significantly predicted by geographic and climate variations, while those of section Cyclobalanopsis were poorly predictable by geographic or climatic diversity. Apart from the different historical environmental changes experienced by different sections, variation of their ecological or climatic tolerances and physiological traits induced varying responses to similar environment changes, resulting in distinct spatial genetic patterns.

Low number of fixed somatic mutations in a long-lived oak tree.

Because plants do not possess a defined germline, deleterious somatic mutations can be passed to gametes, and a large number of cell divisions separating zygote from gamete formation may lead to many mutations in long-lived plants. We sequenced the genome of two terminal branches of a 234-year-old oak tree and found several fixed somatic single-nucleotide variants whose sequential appearance in the tree could be traced along nested sectors of younger branches. Our data suggest that stem cells of shoot meristems in trees are robustly protected from the accumulation of mutations.

Cell-level anatomical characteristics explain high mesophyll conductance and photosynthetic capacity in sclerophyllous Mediterranean oaks.

Leaf mass per area (LMA) has been suggested to negatively affect the mesophyll conductance to CO2 (gm ), which is the most limiting factor for area-based photosynthesis (AN ) in many Mediterranean sclerophyll species. However, despite their high LMA, these species have similar AN to plants from other biomes. Variations in other leaf anatomical traits, such as mesophyll and chloroplast surface area exposed to intercellular air space (Sm /S and Sc /S), may offset the restrictions imposed by high LMA in gm and AN in these species. Seven sclerophyllous Mediterranean oaks from Europe/North Africa and North America with contrasting LMA were compared in terms of morphological, anatomical and photosynthetic traits. Mediterranean oaks showed specific differences in AN that go beyond the common morphological leaf traits reported for these species (reduced leaf area and thick leaves). These variations resulted mainly from the differences in gm , the most limiting factor for carbon assimilation in these species. Species with higher AN showed increased Sc /S, which implies increased gm without changes in stomatal conductance. The occurrence of this anatomical adaptation at the cell level allowed evergreen oaks to reach AN values comparable to congeneric deciduous species despite their higher LMA.

Bidirectional anatomical effects in a mistletoe-host relationship: Psittacanthus schiedeanus mistletoe and its hosts Liquidambar styraciflua and Quercus germana.

PREMISE OF THE STUDY: During the interactions between a parasitic plant and its host, the parasite affects its host morphologically, anatomically, and physiologically, yet there has been little focus on the effect of hosts on the parasite. Here, the functional interactions between the hemiparasitic mistletoe Psittacanthus schiedeanus and its hosts Liquidambar styraciflua and Quercus germana were interpreted based on the anatomical features of the vascular tissues. METHODS: Using standard techniques for light and transmission electron microscopy, we studied the effects of P. schiedeanus on the phloem anatomy of Liquidambar styraciflua and Quercus germana and vice versa. KEY RESULTS: The phloem of P. schiedeanus has larger sieve elements, companion cells, and sieve plate areas when it is parasitizing L. styraciflua than Q. germana; however, the parasite produces systemic effects on the phloem of its hosts, reducing the size of phloem in L. styraciflua but increasing it in Q. germana. Those seem to be the bidirectional effects. No direct connections between the secondary phloem of the parasite and that of its hosts were observed. Parenchymatic cells of L. styraciflua in contact with connective parenchyma cells of the parasite develop half-plasmodesmata, while those of Q. germana do not. CONCLUSIONS: The bidirectional effects between the parasite and its hosts comprise modifications in secondary phloem that are potentially affected by the phenology of its hosts, a combination of hormonal agents such as auxins, and the symplasmic or apoplasmic pathway for solutes import.

Application of Optical Topometry to Analysis of the Plant Epidermis.

The plant epidermis regulates key physiological functions contributing to photosynthetic rate, plant productivity, and ecosystem stability. Yet, quantitative characterization of this interface between a plant and its aerial environment is laborious and destructive with current techniques, making large-scale characterization of epidermal cell parameters impractical. Here, we present our exploration of optical topometry (OT) for the analysis of plant organ surfaces. OT is a mature, confocal microscopy-based implementation of surface metrology that generates nanometer-scale digital characterizations of any surface. We report epidermal analyses in Arabidopsis (Arabidopsis thaliana) and other species as well as dried herbarium specimens and fossilized plants. We evaluate the technology's analytical potential for identifying an array of epidermal characters, including cell type distributions, variation in cell morphology and stomatal depth, differentiation of herbarium specimens, and real-time deformations in living tissue following detachment. As applied to plant material, OT is very fast and nondestructive, yielding richly mineable data sets describing living tissues and rendering a variety of their characteristics accessible for statistical, quantitative genetic, and structural analysis.

Ex situ reconstitution of the plant biopolyester suberin as a film.

Biopolymers often have unique properties of considerable interest as a basis for new materials. It is however not evident how to extract them from plants without destroying their chemical skeleton and inherent properties. Here we report the ex situ reconstitution of the biopolyester suberin as a new waterproof and antimicrobial material. In plant cell walls, suberin, a cross-linked network of aromatic and aliphatic monomers, builds up a hydrophobic protective and antimicrobial barrier. Recently we succeeded in extracting suberin from the plant cell wall using the ionic liquid cholinium hexanoate. During extraction the native three-dimensional structure of suberin was partially preserved. In this study, we demonstrate that this preservation is the key for its ex situ reconstitution. Without any chemical additives or purification, the suberin composing macromolecules undergo self-association on the casting surface forming a film. Suberin films obtained show barrier properties similar to those of the suberin barrier in plants, including a potentially broad bactericidal effect.

Investigating Aspergillus nidulans secretome during colonisation of cork cell walls.

Cork, the outer bark of Quercus suber, shows a unique compositional structure, a set of remarkable properties, including high recalcitrance. Cork colonisation by Ascomycota remains largely overlooked. Herein, Aspergillus nidulans secretome on cork was analysed (2DE). Proteomic data were further complemented by microscopic (SEM) and spectroscopic (ATR-FTIR) evaluation of the colonised substrate and by targeted analysis of lignin degradation compounds (UPLC-HRMS). Data showed that the fungus formed an intricate network of hyphae around the cork cell walls, which enabled polysaccharides and lignin superficial degradation, but probably not of suberin. The degradation of polysaccharides was suggested by the identification of few polysaccharide degrading enzymes (ß-glucosidases and endo-1,5-α-l-arabinosidase). Lignin degradation, which likely evolved throughout a Fenton-like mechanism relying on the activity of alcohol oxidases, was supported by the identification of small aromatic compounds (e.g. cinnamic acid and veratrylaldehyde) and of several putative high molecular weight lignin degradation products. In addition, cork recalcitrance was corroborated by the identification of several protein species which are associated with autolysis. Finally, stringent comparative proteomics revealed that A. nidulans colonisation of cork and wood share a common set of enzymatic mechanisms. However the higher polysaccharide accessibility in cork might explain the increase of ß-glucosidase in cork secretome. BIOLOGICAL SIGNIFICANCE: Cork degradation by fungi remains largely overlook. Herein we aimed at understanding how A. nidulans colonise cork cell walls and how this relates to wood colonisation. To address this, the protein species consistently present in the secretome were analysed, as well as major alterations occurring in the substrate, including lignin degradation compounds being released. The obtained data demonstrate that this fungus has superficially attacked the cork cell walls apparently by using both enzymatic and Fenton-like reactions. Only a few polysaccharide degrading enzymes could be detected in the secretome which was dominated by protein species associated with autolysis. Lignin degradation was corroborated by the identification of some degradation products, but the suberin barrier in the cell wall remained virtually intact. Comparative proteomics revealed that cork and wood colonisation share a common set of enzymatic mechanisms.

UV-laser-based microscopic dissection of tree rings - a novel sampling tool for δ(13) C and δ(18) O studies.

UV-laser-based microscopic systems were utilized to dissect and sample organic tissue for stable isotope measurements from thin wood cross-sections. We tested UV-laser-based microscopic tissue dissection in practice for high-resolution isotopic analyses (δ(13) C/δ(18) O) on thin cross-sections from different tree species. The method allows serial isolation of tissue of any shape and from millimetre down to micrometre scales. On-screen pre-defined areas of interest were automatically dissected and collected for mass spectrometric analysis. Three examples of high-resolution isotopic analyses revealed that: in comparison to δ(13) C of xylem cells, woody ray parenchyma of deciduous trees have the same year-to-year variability, but reveal offsets that are opposite in sign depending on whether wholewood or cellulose is considered; high-resolution tree-ring δ(18) O profiles of Indonesian teak reflect monsoonal rainfall patterns and are sensitive to rainfall extremes caused by ENSO; and seasonal moisture signals in intra-tree-ring δ(18) O of white pine are weighted by nonlinear intra-annual growth dynamics. The applications demonstrate that the use of UV-laser-based microscopic dissection allows for sampling plant tissue at ultrahigh resolution and unprecedented precision. This new technique facilitates sampling for stable isotope analysis of anatomical plant traits like combined tree eco-physiological, wood anatomical and dendroclimatological studies.

The comparative effectiveness of rodents and dung beetles as local seed dispersers in Mediterranean oak forests.

The process of seed dispersal of many animal-dispersed plants is frequently mediated by a small set of biotic agents. However, the contribution that each of these dispersers makes to the overall recruitment may differ largely, with important ecological and management implications for the population viability and dynamics of the species implied in these interactions. In this paper, we compared the relative contribution of two local guilds of scatter-hoarding animals with contrasting metabolic requirements and foraging behaviours (rodents and dung beetles) to the overall recruitment of two Quercus species co-occurring in the forests of southern Spain. For this purpose, we considered not only the quantity of dispersed seeds but also the quality of the seed dispersal process. The suitability for recruitment of the microhabitats where the seeds were deposited was evaluated in a multi-stage demographic approach. The highest rates of seed handling and predation occurred in those microhabitats located under shrubs, mostly due to the foraging activity of rodents. However, the probability of a seed being successfully cached was higher in microhabitats located beneath a tree canopy as a result of the feeding behaviour of beetles. Rodents and beetles showed remarkable differences in their effectiveness as local acorn dispersers. Quantitatively, rodents were much more important than beetles because they dispersed the vast majority of acorns. However, they were qualitatively less effective because they consumed a high proportion of them (over 95%), and seeds were mostly dispersed under shrubs, a less suitable microhabitat for short-term recruitment of the two oak species. Our findings demonstrate that certain species of dung beetles (such as Thorectes lusitanicus), despite being quantitatively less important than rodents, can act as effective local seed dispersers of Mediterranean oak species. Changes in the abundance of beetle populations could thus have profound implications for oak recruitment and community dynamics.

Severe winter rings of oak trees (Quercus robur L.) from Central European Russia.

Oak trees were sampled in a flood plain forest in the valley of the Zapadnaya Dvina (Daugava) river (Tver region, Russia). Annual rings of the time period from 1826 to 2010 were studied. Anatomically distinct rings with a stripe of small-sized cells in the innermost part and narrow earlywood vessels located in three to four rows occurred in 1861, 1862, 1929, 1940, 1942, 1956 and 1979. Deviations of earlywood development were associated with the drop of winter temperature below -42 °C. The percentage of severe winter ring (SWR) occurrence depends upon tree age and decreases from 75.6 % in younger specimens (under 41 years old at the time of the severe winter) to 27.1 % in middle-aged ones (from 41 to 80 years) to 3.5 % in trees older than 80 years. Described anatomical features can be used in the reconstruction of severe winter frequency in the past.

Mycorrhization of Quercus robur L., Quercus cerris L. and Corylus avellana L. seedlings with Tuber macrosporum Vittad.

Tuber macrosporum Vittad. is not a common truffle species, but with remarkable organoleptic qualities and much economic interest. After the addition of truffle spore slurry, 30 seedlings of Quercus robur L., Quercus cerris L. and Corylus avellana L. were grown inside a greenhouse for 11 months before evaluation of the mycorrhizal level. Two different potting mixes were used: a natural soil-based potting mix for Q. robur, Q. cerris and C. avellana and a peat-based potting mix for Q. robur. Quercus robur planted in soil potting mix was the most receptive towards the truffle spore inoculum, with a level of formation of T. macrosporum ectomycorrhizas (ECMs) of approximately 14 %, ranging from a minimum of ∼4 % to a maximum of ∼44 % in different seedlings. No T. macrosporum ECMs developed on Q. cerris (soil potting mix) or on Q. robur (peat potting mix), whereas a low percentage of ECMs was detected on only three C. avellana (soil potting mix) seedlings. The fungus Sphaerosporella brunnea (Alb. & Schwein.) Svrcek & Kubicka was also detected as a contaminant on almost half the truffle-inoculated seedlings. A new detailed description of the morphological and anatomical characteristics of T. macrosporum ECMs and their DNA-based verification with species-specific markers were also reported.

Use of cork powder and granules for the adsorption of pollutants: a review.

Cork powder and granules are the major subproducts of the cork industry, one of the leading economic activities in Portugal and other Mediterranean countries. Many applications have been envisaged for this product, from cork stoppers passing through the incorporation in agglomerates and briquettes to the use as an adsorbent in the treatment of gaseous emissions, waters and wastewaters. This paper aims at reviewing the state of the art on the properties of cork and cork powder and their application in adsorption technologies. Cork biomass has been used on its original form as biosorbent for heavy metals and oils, and is also a precursor of activated carbons for the removal of emerging organic pollutants in water and VOCs in the gas phase. Through this literature review, different potential lines of research not yet explored can be more easily identified.

Allocation of 14C assimilated in late spring to tissue and biochemical stem components of cork oak (Quercus suber L.) over the seasons.

Carbon distribution in the stem of 2-year-old cork oak plants was studied by (14)CO(2) pulse labeling in late spring in order to trace the allocation of photoassimilates to tissue and biochemical stem components of cork oak. The fate of (14)C photoassimilated carbon was followed during two periods: the first 72 h (short-term study) and the first 52 weeks (long-term study) after the (14)CO(2) photosynthetic assimilation. The results showed that (14)C allocation to stem tissues was dependent on the time passed since photoassimilation and on the season of the year. In the first 3 h all (14)C was found in the polar extractives. After 3 h, it started to be allocated to other stem fractions. In 1 day, (14)C was allocated mostly to vascular cambium and, to a lesser extent, to primary phloem; no presence of (14)C was recorded for the periderm. However, translocation of (14)C to phellem was observed from 1 week after (14)CO(2) pulse labeling. The phellogen was not completely active in its entire circumference at labeling, unlike the vascular cambium; this was the tissue that accumulated most photoassimilated (14)C at the earliest sampling. The fraction of leaf-assimilated (14)C that was used by the stem peaked at 57% 1 week after (14)CO(2) plant exposure. The time lag between C photoassimilation and suberin accumulation was ∼8 h, but the most active period for suberin accumulation was between 3 and 7 days. Suberin, which represented only 1.77% of the stem weight, acted as a highly effective sink for the carbon photoassimilated in late spring since suberin specific radioactivity was much higher than for any other stem component as early as only 1 week after (14)C plant labeling. This trend was maintained throughout the whole experiment. The examination of microautoradiographs taken over 1 year provided a new method for quantifying xylem growth. Using this approach it was found that there was more secondary xylem growth in late spring than in other times of the year, because the calculated average cell division time was much shorter.

Analysis of BAC end sequences in oak, a keystone forest tree species, providing insight into the composition of its genome.

BACKGROUND: One of the key goals of oak genomics research is to identify genes of adaptive significance. This information may help to improve the conservation of adaptive genetic variation and the management of forests to increase their health and productivity. Deep-coverage large-insert genomic libraries are a crucial tool for attaining this objective. We report herein the construction of a BAC library for Quercus robur, its characterization and an analysis of BAC end sequences. RESULTS: The EcoRI library generated consisted of 92,160 clones, 7% of which had no insert. Levels of chloroplast and mitochondrial contamination were below 3% and 1%, respectively. Mean clone insert size was estimated at 135 kb. The library represents 12 haploid genome equivalents and, the likelihood of finding a particular oak sequence of interest is greater than 99%. Genome coverage was confirmed by PCR screening of the library with 60 unique genetic loci sampled from the genetic linkage map. In total, about 20,000 high-quality BAC end sequences (BESs) were generated by sequencing 15,000 clones. Roughly 5.88% of the combined BAC end sequence length corresponded to known retroelements while ab initio repeat detection methods identified 41 additional repeats. Collectively, characterized and novel repeats account for roughly 8.94% of the genome. Further analysis of the BESs revealed 1,823 putative genes suggesting at least 29,340 genes in the oak genome. BESs were aligned with the genome sequences of Arabidopsis thaliana, Vitis vinifera and Populus trichocarpa. One putative collinear microsyntenic region encoding an alcohol acyl transferase protein was observed between oak and chromosome 2 of V. vinifera. CONCLUSIONS: This BAC library provides a new resource for genomic studies, including SSR marker development, physical mapping, comparative genomics and genome sequencing. BES analysis provided insight into the structure of the oak genome. These sequences will be used in the assembly of a future genome sequence for oak.

Selective detection of crystalline cellulose in plant cell walls with sum-frequency-generation (SFG) vibration spectroscopy.

The selective detection of crystalline cellulose in biomass was demonstrated with sum-frequency-generation (SFG) vibration spectroscopy. SFG is a second-order nonlinear optical response from a system where the optical centrosymmetry is broken. In secondary plant cell walls that contain mostly cellulose, hemicellulose, and lignin with varying concentrations, only certain vibration modes in the crystalline cellulose structure can meet the noninversion symmetry requirements. Thus, SFG can be used to detect and analyze crystalline cellulose selectively in lignocellulosic biomass without extraction of noncellulosic species from biomass or deconvolution of amorphous spectra. The selective detection of crystalline cellulose in lignocellulosic biomass is not readily achievable with other techniques such as XRD, solid-state NMR, IR, and Raman analyses. Therefore, the SFG analysis presents a unique opportunity to reveal the cellulose crystalline structure in lignocellulosic biomass.

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

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

Drought-induced adaptation of the xylem in Scots pine and pubescent oak.

Drought impairs tree growth in the inner-Alpine valleys of Central Europe. We investigated species-specific responses to contrasting water supply, with Scots pine (Pinus sylvestris L.), threatened by drought-induced mortality, and pubescent oak (Quercus pubescens Willd.), showing no connection between drought events and mortality. The two co-occurring tree species were compared, growing either along an open water channel or at a site with naturally dry conditions. In addition, the growth response of Scots pine to a draining of a water channel was studied. We analysed the radial increment for the last 100 years and wood anatomical parameters for the last 45 years. Drought reduced the conduit area of pubescent oak, but increased the radial lumen diameter of the conduits in Scots pine. Both species decreased their radial increment under drought. In Scots pine, radial increment was generally more dependent on water availability than that in pubescent oak. Irrigated trees responded less negatively to high temperature as seen in the increase in the conduit area in pubescent oak and the removal of the limitation of cell division by high temperatures. After irrigation stopped, tree-ring width for Scots pine decreased within 1-year delay, whereas lumen diameter and cell-wall thickness responded with a 4-year delay. Scots pine seemed to optimize the carbon-per-conduit-costs under drought by increasing conduits diameter while decreasing cell numbers. This strategy might lead to a complete loss of tree rings under severe drought and thus to an impairment of water transport. In contrast, in pubescent oak tree-ring width is less affected by summer drought because parts of the earlywood are built in early spring. Thus, pubescent oak might have gradual advantages over pine in today's climate of the inner-Alpine valley.

New data on ectomycorrhizae and soils of the Chinese truffles Tuber pseudoexcavatum and Tuber indicum, and their impact on truffle cultivation.

Chinese truffles serve as a good complement to the market for Tuber melanosporum (Périgord black truffle). However, Chinese truffles could be introduced accidentally or fraudulently into the plantations of Mediterranean truffles, and they could have a negative effect on truffle production and natural ecosystems. The study of Tuber species from China which are commercialized in Europe began 14 years ago. Tuber pseudoexcavatum was proposed as a new species, and this has been validated by some authors based on molecular and phylogenetic studies. We synthesize their ectomycorrhizae using samples from the type collection, and we compare T. pseudoexcavatum and Tuber indicum ectomycorrhizae. The ectomycorrhizae of these species have a morphology which is related to the ectomycorrhizae of T. melanosporum. We provide useful information for the rapid screening of the above-mentioned Chinese truffles ectomycorrhizae, for the quality control of commercial plants mycorrhized with Tuber. Moreover, we analyze the soil tolerance and the host plant affinity of T. pseudoexcavatum and T. indicum, in order to assess the capacity of both Chinese truffles to penetrate T. melanosporum plantations and habitats.

Nondestructive evaluation of cork enclosures using terahertz/millimeter wave spectroscopy and imaging.

Natural cork enclosures, due to their cell structure, composition, and low moisture are fairly transparent to terahertz (THz) and millimeter waves enabling nondestructive evaluation of the cork's surface and interior. It is shown that the attenuation coefficient of the defect-free cork can be modeled with a Mie scattering model in the weakly scattering limit. Contrast in the THz images is a result of enhanced scattering of THz radiation by defects or voids as well as variations in the cork cell structure. The presence of voids, defects, and changes in grain structure can be determined with roughly 100-300 microm resolution.

Changes in petiole hydraulic properties and leaf water flow in birch and oak saplings in a CO2-enriched atmosphere.

Water relations in woody species are intimately related to xylem hydraulic properties. High CO(2) concentrations ([CO(2)]) generally decrease transpiration and stomatal conductance (g(s)), but there is little information about the effect of atmospheric [CO(2)] on xylem hydraulic properties. To determine the relationship between water flow and hydraulic structure at high [CO(2)], we investigated responses of sun and shade leaves of 4-year-old saplings of diffuse-porous Betula maximowicziana Regel and ring-porous Quercus mongolica Fisch. ex Ledeb. ssp. crispula (Blume) Menitsky grown on fertile brown forest soil or infertile volcanic ash soil and exposed to 500 micromol CO(2) mol(-1) for 3 years. Regardless of species and soil type, elevated [CO(2)] consistently decreased water flow (i.e., g(s) and leaf-specific hydraulic conductivity) and total vessel area of the petiole in sun leaves; however, it had no effect on these parameters in shade leaves, perhaps because g(s) of shade leaves was already low. Changes in water flow at elevated [CO(2)] were associated with changes in petiole hydraulic properties.