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A better understanding of responses to water limitation in woody species can help us to cope with the consequences of the progressing climate change. We focused on the putative transgenerational effects of water limitation in the maternal environment during reproduction. Water was restricted for cuttings of Frangula alnus Mill. in a common garden setting, with a Belgian (local), Italian and Swedish provenance, during the growing season of 2020 and mature berries were collected during the whole reproductive phase. Stones that were extracted from the berries were given a cold stratification. In the next spring, the germination percentage of the stones from the water restricted maternal environment was significantly higher than that of the stones from the normal maternal environment, for the three provenances, notwithstanding the fact that stone weight was not different. The timing of seedling emergence was advanced for the water-limited maternal environment, but only for the stones harvested when mature berry production was the highest (9th and 16th of July 2020). Population differentiation was observed for the timing of seedling emergence, which reflected population differentiation for bud burst of the mother plants in the common garden, including a counter-gradient effect for the Swedish provenance, and corroborating the suggestion that the timing of seedling emergence and leaf phenology may have a common genetic basis. In addition, the Swedish provenance displayed a somewhat more stable germination percentage over the whole berry collection period when the stones were harvested. A partitioning of variance analysis suggested that germination percentage is more genetically determined than timing of seedling emergence, probably reflecting the more important need to sense the environment for an adequate timing of emergence.

Climate change increases global average temperatures worldwide. We tested whether temperature during seed maturation in a broadleaved woody perennial may influence the phenological performance of the seedlings. We performed two controlled crosses of Prunus padus L. in two subsequent years (2015 and 2016). Clonal mother shrubs were subjected to a cold and a warm condition during seed maturation. In the first year after germination, the seedlings from the warm seed maturation condition burst their buds earlier compared with the cold condition seedlings. In contrast, in the second and third years, these seedlings burst their buds later. A temporary maternal effect may have advanced bud burst for the warm condition seedlings in the first year, whereas a delay of bud burst in the following years may be caused by a transgenerational epigenetic memory, putatively expressing a stress reaction upon the suboptimal elevated temperature during seed maturation. A warm spring treatment in 2020 enlarged the difference in timing of bud burst between the cold and warm seed maturation conditions in offspring of both crosses, suggesting that the epigenetic memory is more strongly expressed in a warmer spring environment. The timing of the autumnal leaf senescence in the seedlings was not influenced by the temperature during seed maturation in all observation years, suggesting that autumnal senescence is less (epi)genetically determined compared with bud burst and more sensitive to ambient temperatures.

Longer periods of intensified droughts in Western Europe are predicted due to ongoing climate change. Studying the responses of woody species during intense drought events can help toward understanding the consequences for forest ecosystems. We studied the effects of an intense summer water limitation on several reproductive traits, two years after the treatment, in Frangula alnus Mill. shrubs grown in a common garden. Drought-treated shrubs produced more berries one and two years after the drought event, while the height increment of the second post-treatment year was still significantly retarded. The mean weight of stones from berries picked two years after the drought treatment and their germination percentage, which was corrected for mean stone weight, were higher for the treated shrubs. These results indicate a resource re-allocation toward reproduction, rather than toward growth, which was still in action two years after the water limitation. The higher germination success, which is a transgenerational effect, and which has already been suggested to be an adaptation to survival in more stressful growth conditions, is also still detectable two years after the severe drought. F. alnus produces mature berries continuously during the whole summer. From the middle of July till the end of August, the counts of mature berries, the mean stone weight and the germination percentage, corrected for mean stone weight, decreased, whereas the timing of seedling emergence, also corrected for stone weight, advanced slightly. The timing of seedling emergence correlated weak but significantly with the timing of bud burst in the mother shrubs, with a variance analysis indicating a stronger genetic control for bud burst in comparison to seedling emergence. Several results corroborated previous findings. Population differentiation in the common garden was observed for mature berry counts and for several phenological traits. In conclusion, longer-term effects of drought on reproductive traits in woody species may add more complexity to the consequences of climate change on tree species distributions and survival of forest ecosystems.

Future predictions of forest ecosystem responses are a challenge, as global temperatures will further rise in the coming decades at an unprecedented rate. The effect of elevated temperature on growth performance and phenology of three Prunus spinosa L. provenances (originating from Belgium, Spain, and Sweden) in a common garden environment was investigated. One-year-old seedlings were grown in greenhouse conditions and exposed to ambient and elevated temperatures in the spring (on average 5.6 °C difference) and in the late summer/autumn of 2018 (on average 1.9 °C difference), while they were kept hydrated, in a factorial design. In the following years, all plants experienced the same growing conditions. Bud burst, leaf senescence, height, and diameter growth were recorded. Height and radial growth were not affected in the year of the treatments (2018) but were enhanced the year after (2019), whereas phenological responses depended on the temperature treatments in the year of the treatments (2018) with little carry-over effects in the succeeding years. Spring warming enhanced more height growth in the succeeding year, whereas summer/autumn warming stimulated more radial growth. Spring warming advanced bud burst and shortened the leaf opening process whereas summer/autumn warming delayed leaf senescence and enlarged the duration of this phenophase. These results can help predict the putative shifts in species composition of future forests and woody landscape elements.

Drought periods during the growing season will increase and intensify in Western Europe due to climate change. To better understand the consequences for woody perennials, we restricted watering of potted cuttings of Frangula alnus Mill. in a common garden setting in Belgium during the growing season of 2020. We focused on the responses of three provenances (Belgian, Italian and Swedish) for several reproductive traits in the year of the water limitation. F. alnus, as a shrub species, bears fruits fast. It can blossom on current season’s growth and can therefore produce ripened berries continuously during several months. The total berry count across the whole growing season was much lower in the water-limited plants, independent of the provenance. The local Belgian provenance produced more ripened berries in total than the Italian and Swedish provenances, both in the water-limited plants as in the control plants. Maximal berry production occurred in July. The control plants from the Belgian and Italian provenances displayed a second lower maximum in August. Mainly the Swedish provenance displayed a clear advancement of the maximum berry production among the water-limited plants in comparison to the control plants. Slight differences were detected in the average stone count per berry and in the average stone weight, with both traits displaying a single maximum through time. The Swedish provenance displayed the highest average stone count per berry and the Belgian provenance had the lightest average stone weight, both likely attributable to local adaptation. Remarkably, both stone traits were not affected by the water limitation. Results are discussed in the context of several drought response mechanisms including drought escape, drought avoidance, compensation growth, growth/reproduction trade-off and seed size/number trade-off.

Understanding species-specific responses to climate change allows a better assessment of the possible impact of global warming on forest growth. We studied the responses of the shrub species Corylus avellana L., Corylus maxima Mill. and intermediate forms, together stated as the Corylus taxa, and Cornus sanguinea L. upon periodically elevated temperatures in spring and/or in summer–autumn. Experiments were performed in a common garden, with Belgian and Pyrenean provenances for Corylus avellana and Cornus sanguinea. In the Corylus taxa, a warmer spring resulted in a reduction in height and diameter growth. Remarkably, the reduced diameter increment was restored with full compensation in the following year. The height increment for Cornus sanguinea was larger upon a warmer summer–autumn, concurring with a later leaf senescence. Our results suggest that Corylus is more sensitive to spring warming, influencing growth negatively, whereas Cornus is more sensitive to summer–autumn warming, influencing height growth positively. These deviating responses can be explained, at least partly, by their diverging ecological niches, with the Corylus taxa being more shade-tolerant compared to Cornus sanguinea. The warm spring conditions advanced bud burst in all studied taxa, whereas the warm summer–autumn advanced leaf senescence but prolonged its duration in the Corylus taxa, as well as delayed this phenophase in Cornus sanguinea. Little to no after-effects of the temperature treatments were detected. Although Corylus avellana and Cornus sanguinea plants originated from similar origins, their growth and phenological responses in the common garden diverged, with Corylus being more stable and Cornus displaying more variation between the Belgian and Pyrenean provenances.

Background and Objectives: Studying responses in woody plants upon water limitation is gaining importance due to the predicted increase in frequency and intensity of droughts in Europe. We studied the variation in radial growth and in wood anatomical traits caused by water limited growth conditions in offspring from Quercus petraea (Matt.) Liebl., Q. robur L. and their morphological intermediates grown in the same environment. Materials and Methods: Cross sections were prepared from the stems of 210 three-year-old potted seedlings, comprising control plants and seedlings that experienced from late spring until early autumn of the first growing season two sequential periods of water with-holding each followed by plentiful re-watering. Pith radius, ring width of the three growing seasons and latewood vessel diameter in second and third growing season were measured. Presence of intra-annual density fluctuations, dendritic patterns of latewood vessels and the level of ring closure of earlywood vessels were observed. The traits were modelled to examine the explanatory power of the taxon of the mother tree and the drought treatment. Results: Most of the traits displayed significant differences between offspring from Q. petraea and Q. robur and offspring from the morphological intermediates behaved inconsistent among the traits. Most of the traits were significantly affected by the drought stress in the first growing season. Apart from radial growth, also latewood vessel size was reduced in the two growing seasons following the year in which drought was imposed on the seedlings, suggesting an adaptation to improve the tolerance to drought stress. We also found an indication for a compensation growth mechanism, counteracting the lost growing time during the drought stress, as the level of ring closure of the earlywood vessels in the year following the drought treatment was further advanced in the treated seedlings, an effect that disappeared in the subsequent year. Conclusion: Oaks exposed to drought adapt their growth and xylem structure to improve drought resistance. While youth growth of Q. robur is more competition-oriented, with a faster juvenile growth, Q. petraea seems to invest more in a precautious growth, being more prepared for stressful conditions. It is therefore possible that Q. robur seedlings may suffer more from intensified droughts than Q. petraea seedlings.

Climate change will intensify drought periods during the growing season in Western Europe. We mimicked this prediction by withholding water in summer from young rooted cuttings of Frangula alnus Mill., a common shrub species, originating from different latitudes in Europe (Italy, Belgium and Sweden) and growing in a common garden environment in Belgium. We followed the responses to the drought up to two years after the treatment. Counter-intuitively, the Italian provenance displayed earlier symptoms and stronger effects of water limitation than the other two provenances. A putative higher transpiration in this provenance could be suggested based on a relative larger shoot growth, larger leaves and a higher stomatal density. After the post-drought re-watering, the droughted plants of the Italian provenance entered leaf senescence later than the control plants, likely as a compensation mechanism for the lost growing time. Bud burst in the first year after the drought treatment and leaf senescence in the next autumn were both advanced in the drought treated group when compared with the control plants. Bud burst in the second year after the drought treatment did not display any differentiation anymore between control and drought treated plants. Growth traits also displayed legacies of the water limitation. For example, the drought treated plants showed a lower number of reshoots upon pruning in the year after the drought treatment. Our results suggest that assisted migration from southern Europe to western Europe as a climate change adaptation strategy might not always follow the expected patterns.

Background and Objectives: Summer droughts are expected to increase in central and western Europe both in terms of frequency and intensity, justifying studies on longer term legacies of drought stress on tree species and their hybrids. Materials and Methods: We studied the longer-term after effects of water withholding and re-watering in the first growing season of potted seedlings from the sympatric species Quercus robur L., Q. petraea (Matt.) Liebl. and their morphological intermediates. Phenology, growth, and plant architecture were examined after a cut-back of the stems at the end of the third growing season. Results: The legacy of the first-year water limitation is faded in the phenological response. Nonetheless, leaf senescence occurred later in offspring from Q. robur than in offspring from Q. petraea at the end of the fourth growing season and leafing out tended to be later in the subsequent growing season. Offspring from the intermediate forms displayed variable phenological responses. Height and radial growth were still affected by the drought stress in a taxon-dependent way, with the offspring from Q. petraea displaying growth reduction both in height and diameter, whereas offspring from Q. robur did not show any differences anymore between control and treated plants, demonstrating better post-stress recovery in Q. robur. Offspring from morphological intermediates responded again in a variable way. Although the number of reshoots after cutting back the stems was not affected anymore by the drought treatment in the first growing season, the number of side shoots on the reshoots was still reduced in the drought treated group of plants, independent of the taxon of the mother tree. Conclusions: Together, our results demonstrate the longer-lasting effects of drought stress on oak saplings with regard to growth and plant architecture, with the first being taxon dependent.

© 2019 by the authors. Phenology in perennial plants implies the temporal occurrence of biological events throughout the year. Heritable phenotypic plasticity in the timing of the phenophases can be of importance in the adaptation of woody species to a quickly changing environment. We observed the timing of bud burst, flower opening, leaf senescence and leaf fall in two successive years in a common garden of Crataegus monogyna Jacq. in Belgium, consisting of six local and five non-local provenances. Data were processed with cumulative logistic mixed models. Strong auto-correlation was present among the spring phenophases as well as among the autumnal phenophases, with spring phenophases being negatively correlated with fall phenophases. The strongest between-provenance differentiation was found for the timing of bud burst in spring, followed by flower opening and finally by leaf senescence and leaf fall. Warmer spring temperatures in March 2017 advanced the timing of bud burst, and to a lesser extent of flower opening, in all provenances compared to 2016. However, the advancement was non-linear among the provenances, with the lower latitude provenances being relatively less early and the higher elevation provenances being more late than the local provenances in this year. It can be hypothesized that non-local provenances display larger temporal phenotypic plastic responses in the timing of their spring phenophases compared to local provenances when temperatures in the common garden deviate more from their home-sites. ispartof: FORESTS vol:10 issue:4 status: published

Natural populations of woody species are adapted to local growth conditions. Non-local provenances may sense the difference between the local environment and the site conditions at their origin. To study the impact of provenance selection of planting stock in (re)forestation and landscape plantings, the variation in the adaptive traits bud burst, bud set, flower opening, leaf morphology and height increment was examined within and between local and non-local provenances of Prunus spinosa in a provenance trial, located in Flanders (northern part of Belgium), during two consecutive years (shrubs were three and four years old) which contrasted strongly in spring temperatures. Five Flemish provenances were included together with three commercially available provenances originating from Wallonia (southern part of Belgium), Hungary and Spain. When comparing the two observational years, the Spanish provenance showed a plastic temporal response in bud burst, while the Hungarian provenance and to a lesser extent the Walloon provenance responded plastically in bud set. The Hungarian provenance flushed first in 2013 but was not the first to flush in 2014, whereas in 2014 flowers opened first. The rank order among the provenances in flushing was not maintained in flower opening although both phenophases occur congruently. Wide-leaved shrubs tended to flush and flower slightly earlier and to grow slightly quicker compared to narrow-leaved shrubs, possibly as a result of historical hybridisation processes. Height increment did not vary significantly between the Flemish and commercial provenances, except the Hungarian provenance, which showed a slightly retarded youth growth. The different responses between local and non-local provenances may indicate maladaptation of the latter.

Several phenological phases mark the seasonal growth pattern in temperate woody perennials. To gain further insight into the way these phases react on an altering growth environment, we tested whether vegetative and reproductive phenophases in a shrub species respond differentially among different genetic entities and between two different planting sites. We scored leaf bud burst, flower opening, leaf senescence and leaf fall on 267 ramets of Prunus padus L. belonging to 53 genotypes that were sampled in 9 local populations, and that were planted in 2 common gardens in the northern part of Belgium. The data were processed with cumulative logistic regression. The contribution of genetic and non-genetic components to the total variability varied between the four studied seasonal phenophases. The timing of flower opening displayed the smallest relative amount of intragenotypic variance (between ramets), suggesting a stronger genetic control and a lesser need at the individual plant level for plastic fine tuning to the micro-environment. In addition, whereas leaf bud burst showed the highest relative variance at the interpopulation level among all phenophases, probably at least partly attributable to local adaptation, flower opening displayed the highest intergenotypic variance, which may have been promoted more by assortative mating. Spring phenophases were strongly correlated (r = 0.89) as well as the autumnal phenophases (r = 0.72). Flower opening was not correlated with the autumnal phenophases. Timing of leaf bud burst and leaf senescence were negatively correlated, demonstrating that the length of the growing season enlarged or diminished among the studied genotypes. Although the two planting sites were only 24 km apart, all phenophases were advanced at the less exposed site, indicating a phenotypic plastic response. Together, our results suggest that in P. padus, flowering is less sensitive to environmental variation than leaf bud break and may show a lesser impact of a changing environment on this reproductive phenophase.

In the absence of genetic diversity, plants rely on the capacity of phenotypic plasticity to cope with shifts in environmental conditions. Understanding the mechanisms behind phenotypic plasticity and how local phenotypic adjustments are transferred to clonal offspring, will provide insight into its ecological and evolutionary significance. Epigenetic changes have recently been proposed to play a crucial role in rapid environmental adaptation. While the contribution of epigenetic changes to phenotypic plasticity has been extensively studied in sexual reproducing model organisms, little work has been done on vegetative generations of asexual reproducing plant species. We studied the variability of DNA methylation and bud set phenology of the Lombardy poplar (Populus nigra cv. Italica Duroi), a cultivated tree representing a single genotype worldwide distributed since the eighteenth century. Bud set observations and CpG methyl polymorphisms were studied on vegetative offspring resulting from cuttings grown for one season in a common glasshouse environment. The cuttings were collected from 60 adult Lombardy poplars growing in different environments. The physiological condition of the cuttings was determined by measuring weight and nutrient condition. Methylation sensitive amplified polymorphisms were used to obtain global patterns of DNA methylation. Using logistic regression models, we investigated correlations among epigenotype, bud phenology, and the climate at the home site of the donor trees, while accounting for physiological effects. We found significant epigenetic variation as well as significant variation in bud phenology, in the absence of genetic variation. Remarkably, phenology of bud set observed at the end of the growing season in the common environment was significantly correlated with climate variables at the home site of the mother trees, specifically the average temperature of January and monthly potential evapotranspiration. Although we could not directly detect significant effects of epigenetic variation on phenology, our results suggest that, in the Lombardy poplar, epigenetic marks contribute to the variation of phenotypic response that can be transferred onto asexually reproduced offspring resulting in locally adapted ecotypes. This contributes to the growing evidence that epigenetic-based transgenerational inheritance might be relevant for adaptation and evolution in contrasting or rapidly changing environments.

The maternal temperature during seed development can significantly affect seed dormancy, germination and seedling performance. While the response of germination and seedling phenology to maternal temperatures has been well studied for annuals and conifers, very few studies focus on deciduous trees. To understand the responses of seedlings to variation in maternal temperature during seed maturation, we assessed the germination, bud phenology (bud burst, bud set) and height of full sib families in a common garden. We performed three controlled crosses between three different pairs of genotypes of European black poplar (Populus nigra) to achieve full sib families in three experiments in warm (+ 10 degrees C) and cold (control) maternal environments during crossing and seed maturation. Warmer (+ 10 degrees C) maternal temperatures decreased the seed germination success. The seedlings from the warmer maternal environment also displayed later bud burst and earlier bud set, but only in one out of the three crossings (Proven female x Horrues male). Our results indicate that the maternal environment can considerably impact on seed germination and the phenological responses of even two-year old seedlings suggesting the existence of a memory of maternal temperature during seed maturation. The seedlings resulting from the colder maternal environment grew taller than those from the warmer environment during the first, but not second, growing season. Our results further our understanding of the responses of deciduous forest trees to rapid climate change, but more research is needed to better understand the mechanisms behind the observed effects of maternal warming.

Ulmus laevis Pallas is a rare and endangered tree species in the northern part of Belgium, restricted to a minimal amount of natural populations that mainly consist of a strongly reduced number of individuals. Probably isolated for several generations, random phenotypic variation in the strongly declined populations can be hypothesised. We analysed U. laevis trees grown in a field trial with a single-tree-plot design (completely randomised). The plants were vegetatively propagated through cuttings from nearly all known natural relict populations in Flanders. We analysed three short-shoot leaves from different ramets from different genotypes from eleven natural populations. Leaf size and shape variables were computed using landmark and procruste methods. We visualised the variability in leaf morphology in a principal component analysis (PCA) including shape, size, and categorical morphological variables. The variance structure of the morphological variables was studied applying mixed modelling methods. The overall PCA distinguished two deviating natural populations. The Zandhoven population showed a deviating simple leaf margin toothing, correlated with larger numbers of split secondary veins. The Halle population is characterised by absence of pubescence at the lower side of the leaves. Both relict populations with deviating morphological leaf characteristics may point to an ecotype evolution putatively due to isolation combined with greatly diminished population sizes. The variance structure analysis of individual leaf variables showed that the deviating categorical leaf characters are under stronger genetic control (specialist characters) compared to the more plastic variables.

Flushing in spring marks an important adaptive process in a tree’s growth cycle. We studied bud burst in three provenances of a common small tree Frangula alnus Mill., originating from Italy, Belgium and Sweden. We observed timing of bud burst and leaf senescence in a common garden, and timing and duration of bud burst in greenhouse conditions (cuttings) with different temperature regimes, all located in Belgium. The early bud burst response of the southern European provenance together with the later leaf senescence compared to the local provenance indicated population differentiation that may, at least partly, be driven by local adaptation to a longer growing season. The duration of the process was longer in the cold greenhouse compared to the local provenance, whereas it responded similar as the local provenance in the warm greenhouse, suggesting adaptation to warmer conditions. Unexpectedly, the northern European provenance expressed a plastic reaction to warmer temperatures in the field trial and in the greenhouse conditions flushing in both cases earlier than the local provenance. The duration of the process in the warm greenhouse compared to the local provenance was shorter, whereas in the cold greenhouse it was similar. This result again suggested that not only the onset, but also the duration of the bud burst process was a plastic reaction to the warmer conditions compared to its site of origin. Together, our results suggest two mechanisms driving bud burst in F. alnus provenances, a co-gradient and a counter-gradient variation depending on the latitude of origin.

Spontaneous hybrids in natural regenerations of A. glutinosa cannot be excluded if A. incana is occurring in the neighbourhood, and has to be taken into account when restoring swamp forests. Swamp forests with A. glutinosa are endangered in Flanders due to habitat destruction and fragmentation, and therefore restoration is promoted. At the other hand, frequent plantations of A. incana, a non-native species in Flanders (northern part of Belgium), may facilitate spontaneous hybrids between the two species as they are interfertile. We assessed phenotypic variability of both species and putative hybrids using nine leaf, five infructescence and eight bud characters, and additionally height growth. We focussed on a field trial established with seeds collected in fifteen autochthonous A. glutinosa stands and on A. incana trees of unknown origin sampled at five locations in Flanders. We applied geometric morphometric methods, principal component analyses and mixed modelling methods. We found three individuals with intermediate morphological traits between A. glutinosa and A. incana among the eleven pedigrees in one of the two natural A. glutinosa populations where also planted A. incana was present. Their height growth was variable and only one of the three fructified, with seeds showing a normal germination success. These individuals can be hybrids with intermediate features between the parental species or they may display the natural variability of the latter with overlapping traits. Other individuals with intermediate leaf shape but less intermediate features for other leaf, bud and infructescence characters, found in the pedigrees of additional four A. glutinosa populations, may point to the relative wide natural variability for leaf shape within the parental species. Alternatively, these may concern backcrossed individuals or matro/patroclinal inheritance in hybrids. An inter-population variance study showed that several leaf characters are under a relative genetic control, underlining the importance of vegetative organs in taxonomic treatment of species complexes. The occurrence of spontaneous hybrids in natural regenerations of A. glutinosa cannot be excluded if A. incana is present in the neighbourhood, and has to be taken into account both in seed collections for the creation of autochthonous planting stock as when restoring swamp forests.

The evaluation of the molecular signatures of selection in species lacking an available closely related reference genome remains challenging, yet it may provide valuable fundamental insights into the capacity of populations to respond to environmental cues. We screened 25 native populations of the tree species Frangula alnus subsp. alnus (Rhamnaceae), covering three different geographical scales, for 183 annotated single-nucleotide polymorphisms (SNPs). Standard population genomic outlier screens were combined with individual-based and multivariate landscape genomic approaches to examine the strength of selection relative to neutral processes in shaping genomic variation, and to identify the main environmental agents driving selection. Our results demonstrate a more distinct signature of selection with increasing geographical distance, as indicated by the proportion of SNPs (i) showing exceptional patterns of genetic diversity and differentiation (outliers) and (ii) associated with climate. Both temperature and precipitation have an important role as selective agents in shaping adaptive genomic differentiation in F. alnus subsp. alnus, although their relative importance differed among spatial scales. At the 'intermediate' and 'regional' scales, where limited genetic clustering and high population diversity were observed, some indications of natural selection may suggest a major role for gene flow in safeguarding adaptability. High genetic diversity at loci under selection in particular, indicated considerable adaptive potential, which may nevertheless be compromised by the combined effects of climate change and habitat fragmentation. ispartof: Heredity vol:115 issue:5 pages:115-425 ispartof: location:England status: published

To study the impact of provenance selection of planting stock in (re)forestation and landscape plantings, the variation in bud and flower phenology, height increment and shrub morphology was examined within and between the local and non-local provenances of Crataegus monogyna in a provenance trial. Seven Belgian provenances were included, five in Flanders and two in Wallonia, completed with three commercial provenances originating from Italy, Hungary and the United Kingdom. A completely randomized design (single tree plots) was adopted. Data were processed using mixed modelling techniques. South European provenances flushed up to 7 days earlier than the Flemish provenances, whereas the Walloon provenances flushed 4 days later. Compared to the Flemish provenances, Southern provenances and Walloon provenances flowered 4 days earlier and 2 days later respectively. Height increment and shrub morphology did not vary significantly between the Belgian and commercial provenances. Bud burst varied less than flowering among the Flemish and the Walloon provenances. This possibly indicates a higher selection pressure on bud burst than on flowering. The higher time lag between the different provenances for bud burst in comparison to flowering indicates a stronger plastic response for the latter. The results point out the importance of bud burst as an indicator of local adaptation to geo-climatologic components. Finally, it appeared that old hedges are likely to be good sources of locally adapted seed.

Prunus spinosa is a highly esteemed shrub in forest and landscape plantings. Shrubs with larger organs occur often and are considered either as large fruited forms of P. spinosa or as P. x fruticans, involving a hybridization process with the ancient cultivated P. insititia (crop-to-wild gene flow). As climate change may augment hybridization processes in the future, a hybrid origin is important to detect. In addition, studying crop-to-wild gene flow can give insights in putative consequences for the wild populations. We studied the P. spinosa–P. x fruticans group, focusing on morphology and phenology in three experimental plantations. Two plantings harbored cuttings of P. spinosa (clone plantations). A third plantation comprised of a half-sib offspring from a population with both P. spinosa and P. x fruticans (family plantation). Several results point to a hybridization process as the origin of P. x fruticans. The clone plantation revealed endocarp traits to be more genetically controlled than fruit size, while this was the opposite in the family plantation, suggesting the control of fruit size being derived from the putative P. insititia parent. Bud burst, flower opening, and leaf fall were genetically controlled in the clone plantation, whereas in the family plantation intrafamily variability was remarkably large for the bud burst and leaf fall, but not for the flower opening. This suggests there is a reduced genetic control for the first two phenophases, possibly caused by historic hybridization events. Pubescence on the long shoot leaves in the family plantation deviated from the short shoot leaves on the same plants and from long and short shoot leaves in the clone plantation, suggesting again a P. insititia origin. Finally, we quantified spatial phenotypic plasticity, indicating how P. spinosa may react in a changing environment. In contrast to the bud burst and leaf fall, flower opening did not demonstrate plasticity. The fruit size was diminished at the growth site with the shortest growing season while interestingly, the leaf width was enlarged. Leaf size traits appeared more plastic on the long shoots compared to the short shoots, although partitioning of variance did not display a lesser genetic control.

More severe summer droughts are predicted for mid-latitudes in Europe. To evaluate the impact on forest ecosystems and more specifically on forest regeneration, we studied the response to summer drought in oak seedlings (Quercus petraea). Acorns were collected from different mother trees in three stands in Belgium, sown in pots and grown in non-heated greenhouse conditions. We imposed severe drought on the seedlings in early summer by first watering the pots to saturation and then stopping any watering. Weight of the pots and stomatal conductance were regularly measured. Re-watering followed this drought period of five weeks. Height of the seedlings and apical bud development were observed. Stomatal resistance increased towards the end of the experiment in the drought-treated group and was restored after re-watering. The seedlings from the drought treatment displayed a higher probability to produce additional shoot growth after re-watering (p ≤ 0.05). A higher competition for resources (two plants per pot) increased this chance. Although this chance was also higher for smaller seedlings, the actual length of the extra growth after re-watering was higher for larger seedlings (p ≤ 0.01). Both in the drought-treated and in the control group the autochthonous provenance growing on a xeric site produced less extra shoots compared to the two other provenances. Finally, stressed plants showed less developed apical buds compared to the control group after re-watering, suggesting a phenological effect on the growth cycle of oaks (p ≤ 0.0001). The higher chance for an extra shoot growth after the drought period can be considered as a compensation for the induced growth arrest during the drought period.

Despite their genetic homogeneity, many taxonomic species are described as European dogroses (Rosa L. section Caninae (DC.) Ser.) with consistent morphological variability. Here we report a morphometric study of 27 hip and leaf characters of proximate dogrose populations that include Rosa agrestis, a member of the subsection Rubigineae, in Flanders. In principal components analysis one R. agrestis population in Kanne had intermediate morphology between R. canina and R. corymbifera, on the one hand, and three other R. agrestis populations, on the other, suggesting hybridogenic origin. Half-sib R. agrestis seedlings were grown under controlled conditions and their leaf characters were studied. A tendency toward deviating morphology in seedlings from the Kanne population, analogous to the mother plants in the field, and a correlation for specific leaf characters between mother plants and their corresponding seedlings reinforce a genetic basis for the observed divergence in the Kanne population. The assumed hybridogenic mother plants did not produce fewer seeds per hip than the others of the pure populations. In addition, for all sampled dogroses, which included six species, the diameter of the discus was correlated with the number of seeds per hip, whereas the diameter of the orifice was not correlated with this character. This implies that only the diameter of the orifice and not the diameter of the discus might be decisive for species identification among European dogroses.

Hybrids between Quercus robur and Q. petraea have been a sought topic of many studies in Europe during the last decades. Here, leaf and fruit morphology were studied in five oak stands where both species occur naturally intermixed. The stands are relicts of old, possibly medieval coppice wood. Twenty two leaf characters and nine fruit characters were assessed on three leaves and three fruits per tree and for thirty trees per stand. A principal component analysis (PCA) resulted in a bimodal distribution with restricted overlap along the first component when both leaf and fruit data were processed together. For leaf and fruit data separately, the analysis produced only continuous clusters of trees. Two types of putative hybrids can be defined that either show a petiole length of the leaf (PL) according to Q. robur and a petiole length of the fruit (FP1) according to Q. petraea, or vice versa. These hybrids cluster within both groups of the PCA analysis, but not all are situated close to or in the intermediate area between the groups. A lowered mean relative number of developed acorns in the hybrid groups in comparison to their putative maternal parent, based on the assumption of matroclinal inheritance of PL, is observed. This might indicate a reduced ability for successful fertilisation in the hybrids. These results suggest the presence of putative hybrids and introgressed forms within the morphological distinct Q. robur and Q. petraea groups and argument for a taxonomically defined Q. x rosacea based on PL and FP1 limits.

The response of trees to a changing climate can be affected by transgenerational phenotypic plasticity, i.e. phenotypic variation that is conserved and transferred to the offspring. Transgenerational plasticity that is influenced by epigenetics (heritable changes in gene function that do not result from changes in DNA sequence) during both sexual and asexual reproduction are of major relevance for adaptation of plants to climate change. To understand the transgenerational effects on the responses of vegetatively propagated poplar (Populus deltoides and P. trichocarpa) ramets (cuttings) to a changing environment, we tested whether the temperature and photoperiod experienced by the mother trees (genets) persistently affects the phenology of the cuttings grown in a common environment. We weekly monitored the bud phenology of the cuttings collected from the parent trees that have been growing across Europe along a >2100 km latitudinal gradient for at least 18 years. In addition, we asked whether there was variation in DNA methylation as measured by Methylation Sensitive Amplified Fragment Length Polymorphism (MSAPs) in the clones due to the different environmental conditions experienced by the parent trees. Our results indicate a transgenerational effect on bud phenology in the asexually reproduced offspring (vegetative cuttings). The temperatures experienced by the parent tree clones (from different geographic regions) altered the bud flush of the cuttings in the common garden. However, no significant epigenetic variation was detected in the cuttings of the parent trees within single genotypes growing under different climates. In sum, our results show that trees have the potential to respond to rapid climate change but the mechanism behind these changes needs to be further investigated by more powerful molecular methods like whole-genome bisulphite sequencing techniques.

Rosa arvensis is a naturally rare and scattered indigenous wild rose species in Flanders, the northern part of Belgium. As is the case for many light demanding woody species in this area, it is currently threatened by habitat fragmentation and destruction due to high human pressure. Recent inventories revealed a restricted distribution pattern for this rose, concentrated mainly in two regions of the south western part of Flanders. Surprisingly, strong differentiation was observed among natural populations in these two proximate regions in both an AFLP-based and a morphological analysis. A common garden experiment indicated a partly genetic basis for the morphological divergence. Additionally, the AFLP analysis of roses sampled in the same forested area within one of the two regions resulted in two differentiated gene pools. Possible causes for the observed differentiation can be adaptive divergence, founder effects and/or historical hybridisation with dogroses. Together, the congruent genetic and morphometric differentiation between the two geographic regions urges a cautious approach in conservation programs.

Multivariate analysis of both endocarp and leaf morphometrics is combined with cluster analysis and Bayesian inference of AFLP markers to assess the morphologic and genetic variation of five European members of Prunus section Prunus (P. cerasifera, P. domestica, P. insititia, P. spinosa, and P. × fruticans). Endocarp morphometrics separate most Prunus taxa studied, but overlap remains between P. domestica and P. cerasifera, and P. spinosa and P. × fruticans. Leaf morphometrics yield better separation of P. domestica and P. cerasifera, but do not allow distinction between P. spinosa and P. × fruticans. Both cluster analysis and PCoA of AFLP markers equally produce three distinct clusters. A first consists of all P. cerasifera samples and the sole P. cocomilia; a second cluster includes all individuals of P. domestica and P. insititia; and a third group comprises all P. spinosa and P. × fruticans samples.

†Background and Aims Identification of Prunus groups at subspecies or variety level is complicated by the wide range of variation and morphological transitional states. Knowledge of the degree of variability within and between species is a sine qua non for taxonomists. Here, a detailed study of endocarp dimension and shape variation for taxa of Prunus section Prunus is presented. †Methods The sample size necessary to obtain an estimation of the population mean with a precision of 5 % was determined by iteration. Two cases were considered: (1) the population represents an individual; and (2) the population represents a species. The intra-individual and intraspecific variation of Prunus endocarps was studied by analysing the coefficients of variance for dimension and shape parameters. Morphological variation among taxa was assessed using univariate statistics. The influence of the time of sampling and the level of hydration on endocarp dimensions and shape was examined by means of pairwise t-tests. In total, 14 endocarp characters were examined for five Eurasian plum taxa. †Key Results All linear measurements and index values showed a low or normal variability on the individual and species level. In contrast, the parameter ‘Vertical Asymmetry’ had high coefficients of variance for one or more of the taxa studied. Of all dimension and shape parameters studied, only ‘Triangle’ differed significantly between mature endocarps of P. insititia sampled with a time difference of 1 month. The level of hydration affected endocarp dimensions and shape significantly. †Conclusions Index values and the parameters ‘Perimeter’, ‘Area’, ‘Triangle’, ‘Ellipse’, ‘Circular’ and ‘Rectangular’, based on sample sizes and coefficients of variance, were found to be most appropriate for further taxonomic analysis. However, use of one, single endocarp parameter is not satisfactory for discrimination between Eurasian plum taxa, mainly because of overlapping ranges. Before analysing dried endocarps, full hydration is recommended, as this restores the original dimensions and shape.

Autochthonous genetic resources of woody plants have become seriously endangered in Flanders because of the particularly low and fragmented forest cover, centuries of intensive forest use in this highly populated area and the wide-spread usage of non-autochthonous planting stock in reforestation and landscape plantings. Intraspecific hybridisation between remnant autochthonous populations and foreign genotypes, which can show inadequate adaptation, may influence the autochthonous genetic constitution and fitness in the long term. As several European countries face similar problems, the objective of this paper is to outline the conservation measures that are taken in Flanders. The central aim is to maintain and create the necessary conditions for natural and flexible evolution of the genetic diversity of autochthonous trees and shrubs. An inventory survey to locate remaining autochthonous populations was started in 1997 and will be completed in 2006. Relict populations are preserved in clonal banks. Central issues are the production of autochthonous planting stock through in situ seed collection, the approval of seed sources and stands and the creation of seed orchards. Conservation actions are discussed.

The increasing severity and frequency of summer droughts at mid-latitudes in Europe may impact forest regeneration. We investigated whether the sympatric species Quercus robur L., Q. petraea (Matt.) Liebl., and their morphological intermediates respond differentially to water deficit. Acorns were sourced from a naturally mixed population. Half of the potted seedlings were subjected to two successive drought periods during the first growing season, each followed by a plentiful re-watering. The surviving drought-exposed seedlings subsisted independent of the taxon of the mother tree. The phenological responses were also taxon-independent. However, drought-exposed plants showed a retarded height growth in the year following the treatment which was taxon-dependent. Offspring from Q. robur and from trees with leaves resembling Q. robur leaves and infructescences resembling Q. petraea infructescences showed a stronger decrease in height growth compared to the offspring from Q. petraea and from trees with leaves resembling Q. petraea leaves and infructescences resembling Q. robur infructescences. Diameter growth in the year following the drought treatment showed a weak taxon-dependent response. Together, our results may suggest that the composition of oak species and their hybrids in natural oak forests could be altered upon prolonged periods of precipitation deficit. ispartof: Forests vol:8 issue:11 pages:407-423 status: published

Delineation of seed zones or provenance regions to preserve local adaptation is a common practice in forestry and restoration, as locally adapted plants generally possess relatively high levels of productivity and resistance. Provenance trials typically quantify the degree of phenotypic divergence among individuals and populations raised under common conditions, which is time‐consuming and potentially confounded by phenotypic plasticity and maternal effects. Here, we put forward population genomics, the screening of individual genomes for the genetic signature of adaptation, as a fast and reliable strategy to evaluate seed zone delineation. To illustrate the value of this approach, we quantified the degree of genomic adaptation within and among Belgian black alder Alnus glutinosa provenances and compared results with traditional provenance trials. Distant European reference regions were included to validate the approaches, as larger environmental differences at a European scale are expected to result in larger adaptive responses. Local provenances did not perform better than foreign provenances at the scale of Belgian seed zones, in contrast to the comparisons with the distant European regions. A significant site effect indicated that plastic responses rather than local adaptation explain phenotypic differences among seed zones. The common garden revealed little evidence for adaptation for all measured traits, both among seed zones and among distant regions. The number and strength of genetic outliers was not significantly larger among Belgian seed zones than within these seed zones, but was significantly larger between Belgian seed zones and the distant European reference regions. Synthesis and applications. The lack of adaptive divergence among Belgian seed zones supports an expansion of current provenance regions into larger seed zones. The results also show that population genomics can be an accurate and time‐efficient resource to assist decisions on seed sourcing. This highlights the importance of raising awareness of the potential benefits of this novel approach among policy makers, foresters and restoration practitioners. ispartof: Journal of Applied Ecology vol:51 issue:5 pages:1218-1227 status: published

Interspecific hybridisation and gene flow from cultivated plants may have profound effects on the evolution of wild species. Considering the cultural history and past use of Ulmus minor and Ulmus glabra trees in Flanders (northern Belgium), we investigated the extent of human impact on the genetic variation of the remaining, supposedly indigenous elm populations. We therefore examined the rate of interspecific hybridisation, which is expected to be higher under human influence, the occurrence of clones within and among locations, the presence of cultivars and their possible offspring. Based on results produced using 385 amplified fragment length polymorphic (AFLP) markers, 46 % of the 106 investigated Flemish elms appeared to be F1 hybrids or backcrosses to one of the parent species, while no F2 hybrids (F1×F1 progeny) were found. Clonality was mainly found among U. minor and hybrids, which are more likely to form root suckers or sprouts as opposed to U. glabra. The majority of the studied locations (76 % of the locations with multiple samples) showed evidence of clonal reproduction. Several, sometimes distant, locations shared a multilocus lineage. We also found indications of gene flow from cultivated elms into native species. It is conceivable that reproductive material has been moved around extensively, obscuring the natural genetic structure of the elm populations. The results help guide the Flemish elm genetic resources conservation programme. Interspecific hybridisation and gene flow from cultivated plants may have profound effects on the evolution of wild species. Considering the cultural history and past use of Ulmus minor and Ulmus glabra trees in Flanders (northern Belgium), we investigated the extent of human impact on the genetic variation of the remaining, supposedly indigenous elm populations. We therefore examined the rate of interspecific hybridisation, which is expected to be higher under human influence, the occurrence of clones within and among locations, the presence of cultivars and their possible offspring. Based on results produced using 385 amplified fragment length polymorphic (AFLP) markers, 46 % of the 106 investigated Flemish elms appeared to be F1 hybrids or backcrosses to one of the parent species, while no F2 hybrids (F1 × F1 progeny) were found. Clonality was mainly found among U. minor and hybrids, which are more likely to form root suckers or sprouts as opposed to U. glabra. The majority of the studied locations (76 % of the locations with multiple samples) showed evidence of clonal reproduction. Several, sometimes distant, locations shared a multilocus lineage. We also found indications of gene flow from cultivated elms into native species. It is conceivable that reproductive material has been moved around extensively, obscuring the natural genetic structure of the elm populations. The results help guide the Flemish elm genetic resources conservation programme.

Pinoresinol-lariciresinol and isoflavone reductase classes are phylogenetically related, as is a third, the so-called “isoflavone reductase homologs.” This study establishes the first known catalytic function for the latter, as being able to engender the NADPH-dependent reduction of phenylcoumaran benzylic ethers. Accordingly, all three reductase classes are involved in the biosynthesis of important and related phenylpropanoid-derived plant defense compounds. In this investigation, the phenylcoumaran benzylic ether reductase from the gymnosperm, Pinus taeda, was cloned, with the recombinant protein heterologously expressed inEscherichia coli. The purified enzyme reduces the benzylic ether functionalities of both dehydrodiconiferyl alcohol and dihydrodehydrodiconiferyl alcohol, with a higher affinity for the former, as measured by apparent K m andV max values and observed kinetic3H-isotope effects. It abstracts the 4R-hydride of the required NADPH cofactor in a manner analogous to that of the pinoresinol-lariciresinol reductases and isoflavone reductases. A similar catalytic function was observed for the corresponding recombinant reductase whose gene was cloned from the angiosperm,Populus trichocarpa. Interestingly, both pinoresinol-lariciresinol reductases and isoflavone reductases catalyze enantiospecific conversions, whereas the phenylcoumaran benzylic ether reductase only shows regiospecific discrimination. A possible evolutionary relationship among the three reductase classes is proposed, based on the supposition that phenylcoumaran benzylic ether reductases represent the progenitors of pinoresinol-lariciresinol and isoflavone reductases.

Background and aims – Seed sourcing on a local scale is an emerging awareness among nature conservationists. Guidelines should be tuned for woody species in highly anthropogenic landscapes such as Flanders (Belgium). We investigated the genetic variation within and between eight autochthonous Flemish Prunus spinosa populations and assessed their potential as seed source for gene conservation and ecological restoration. Methods and key results – All sampled sites were old hedges or wooded banks. Using AFLP, the overall gene diversity ( H t ) was estimated at 0.13, with a low average between population diversity ( H b = 0.02) and a comparatively high average within-population diversity ( H w = 0.11). The genetic differentiation was remarkably variable among populations (φ ST ranging from 0.07 to 0.43). There was no clear relationship between genetic and geographic distances, nor between genetic and morphometric characteristics. Only a small but significant correlation between endocarp and leaf morphological characters was observed. Conclusions – The variable genetic differentiation among populations points to different vegetation histories. Two populations, DEF and WES, were characterized by a dominant presence of sloe, high genetic differentiation from all other populations and low within-population diversities. No obvious morphometric variability of leaves could be related to these genetically deviating populations, however, WES showed a deviating endocarp morphology. Possibly, these two populationswere originally planted or supplemented using local and much related material whereas the others may have been planted with more diverse material. The relatively high within-population diversities and moderate, although variable, between-population differentiation of the other Flemish populations point to a considerable amount of gene exchange and can justify extensive seed sourcing for the production of autochthonous planting stock. These results stress the importance of a proper evaluation of genetic diversity for seed source selection and the need for regional field-applicable guidelines.

In Arabidopsis the promoter of the gene encoding S-adenosyl-L-methionine synthetase (SAM-S) Psam-1 confers expression preferentially in the vascular tissue. In search for promoters that drive expression in particular cells of the lignifying tissues in trees, we have analyzed the expression pattern conferred by the Psam-1 promoter in transgenic poplar. Histochemical analyses demonstrated beta-glucuronidase (GUS) activity mainly in phloem and cortex tissue throughout the plant, and in root tips. Fluorimetric assays showed high GUS activity in the tissues outside (phloem, cortex and cork) compared to those inside (xylem and pith) of the cambial layer. In contrast, the endogenous SAM-S activity was high in tissues inside and low in tissues outside of the cambial layer. RNA gel blot analysis demonstrated a high transcript level of the endogenous sam-s gene(s) in tissues both outside and inside the cambial layer. This indicates that the low SAM-S activity in the bark was at least partially due to translational and/or post-translational regulation of the endogenous sam-s gene(s). In dormant transgenics, the tissue specificity was conserved, but the activity levels were up to 10-fold reduced.

Native plant species are routinely planted or sown in ecological restoration projects, but successful establishment and survival depend on where and how seeds are collected. Research suggests that it is important to use locally adapted seeds. Local populations often show a home-site advantage and non-local genotypes may be maladapted to local environmental conditions. Furthermore, intraspecific hybridisation of local and non-local genotypes may have a negative impact on the genetic structure of local populations via mechanisms such as outbreeding depression. Many species show a strong small-scale genetic differentiation between different habitats so that matching habitats of the restoration and donor site can be more important than minimizing geographical separation. It is a challenge to identify appropriate seed sources because strong small-scale population differentiation makes it difficult to delineate geographically defined seed zones to which seed exchange should be limited. Moreover, it is important to consider the genetic diversity of introduced material because it may be crucial to avoid genetic bottlenecks, inbreeding depression and poor establishment of plant populations. Repeated propagation in stock, which is often required to obtain a sufficient amount of seeds, can further reduce genetic diversity and may select for particular genotypes. Negative impacts of improper seed choice for nursery planting stock may become detectable only after many years, especially in long-lived and slow growing plants. Although scientific information on many species remains limited, the increasing demand for translocation of seed means that mandatory regulations are necessary. Guidelines should prescribe a specification of seed provenance, a record of genetic diversity of wild collections and rules for subsequent processing such as direct transfer and propagation of stock or seed orchards. We use a literature review to evaluate current legislation and to develop recommendations for herbaceous and woody species. / Es ist heute gängige Praxis, heimische Pflanzenarten zu Renaturierungszwecken auszubringen. Die erfolgreiche Etablierung hängt jedoch davon ab, wie und wo das Saat- oder Pflanzgut gewonnen wurde. Forschungsergebnisse legen nahe, dass nur lokal angepasste Herkünfte eingesetzt werden sollten. Es ist vielfach festgestellt worden, dass sie eine höhere Fitness als fremde Herkünfte besitzen, und letztere sind daher schlecht an lokale Umweltbedingungen angepasst. Eine intraspezifische Hybridisierung mit gebietsfremden Populationen kann weiterhin einen negativen Einfluss auf die genetische Struktur von lokalen Populationen haben wie z.B. outbreeding depression. Einige Arten zeigen eine starke kleinräumige genetische Differenzierung zwischen verschiedenen Habitaten, so dass die Übereinstimmung von Spender- und Renaturierungsflächen wichtiger sein kann als eine minimale Entfernung. Diese kleinräumige Differenzierung macht es schwierig, geographische Regionen festzulegen, innerhalb derer ein Austausch von Pflanzenmaterial unbedenklich ist. Darüber hinaus muss die genetische Diversität des gesammelten und eingeführten Materials berücksichtigt werden, um genetische Flaschenhälse, Inzuchtdepression und eine geringe Etablierungsrate zu vermeiden. Eine wiederholte Kultivierung und Vermehrung von Pflanzenarten, die oft notwendig ist, um eine ausreichende Samenmenge zu produzieren, kann zu einer weiteren Reduktion der genetischen Diversität beitragen oder zu einer Selektion bestimmter Genotypen führen. Negative Auswirkungen einer ungeeigneten Auswahl von Herkünften werden insbesondere bei langlebigen und langsam wachsenden Arten häufig erst nach vielen Jahren feststellbar. Angesichts des wachsenden Bedarfs an Wildpflanzensaatgut sind verbindliche Regelungen notwendig, auch wenn die Datenlage für viele Arten immer noch unzureichend ist. Diese Richtlinien sollten Vorgaben zur Herkunft und Diversität des Saatgutes bei der Sammlung am natürlichen Standort machen und die nachfolgende Prozesskette von der Saatgutreinigu g und Lagerung über die Vermehrung bis hin zur Ausbringung regeln. Unter Verwendung von Literaturdaten bewerten wir die gegenwärtige Gesetzgebung und entwickeln Empfehlungen sowohl für krautige Pflanzen als auch für Gehölze.

It has previously been shown (D.R. Gang et al., 1999, J Biol Chem 274: 7516–7527) that the most abundant protein in the secondary xylem of poplar (Populus trichocarpa cv. `Trichobel') is a phenylcoumaran benzylic ether reductase (PCBER), an enzyme involved in lignan synthesis. Here, the distribution and abundance of PCBER in poplar was studied at both the RNA and protein level. The cellular expression pattern was determined by immunolocalization of greenhouse-grown plants as well as of a field-grown poplar. Compared to other poplar tissues, PCBER is preferentially produced in the secondary xylem of stems and roots and is associated with the active growth period. The protein is present in all cells of the young differentiating xylem, corresponding to the zone of active phenylpropanoid metabolism and lignification. In addition, PCBER is located in young differentiating phloem fibers, in xylem ray parenchyma, and in xylem parenchyma cells at the growth-ring border. Essentially the same expression pattern was observed in poplars grown in greenhouses and in the field. The synthesis of PCBER in phenylpropanoid-synthesizing tissues was confirmed in a bending experiment. Induction of PCBER was observed in the pith of mechanically bent poplar stems, where phenylpropanoid metabolism is induced. These results indicate that the products of PCBER activity are synthesized mainly in lignifying tissues, suggesting a role in wood development.

Proteins that are preferentially produced in developing xylem may play a substantial role in xylogenesis. To reveal the identity of these proteins, comparative two-dimensional polyacrylamide gel electrophoresis was performed on young differentiating xylem, mature xylem, and bark of poplar (Populus trichocarpa Hook. cv. 'Trichobel') harvested at different times of the year. The most-abundant xylem proteins were identified by microsequence analysis. For 17 of these proteins a putative function could be assigned based on similarity with previously characterized proteins, and for 15 out of these corresponding expressed sequence tags (ESTs) were found in the poplar EST database. The identified xylem-preferential proteins, defined by comparing the protein patterns from xylem and bark, were all involved in the phenylpropanoid pathway: two caffeoyl-coenzyme A O-methyltransferases (CCoAOMT), one phenylcoumaran benzylic ether reductase (PCBER), one bispecific caffeic acid/5-hydroxyferulic acid O-methyltransferase (COMT), five S-adenosyl-L-methionine synthetases, and one homologue of glycine hydroxymethyltransferase (GHMT). Remarkably, the biological function of the two most-abundant xylem-preferential proteins (PCBER and a GHMT homologue) remains unclear. In addition, several housekeeping enzymes were identified: two enolases, two glutamine synthetases, one 70-kDa heat-shock cognate, one calreticulin, and one alpha-tubulin. In comparison to the xylem-preferential proteins, the housekeeping proteins were expressed at significant levels in the bark as well. Also, several additional protein spots were detected for CCoAOMT, PCBER, and COMT by immunoblot. Our data show that for the study of xylogenesis, two-dimensional protein gel comparisons combined with systematic protein sequencing may yield information complementary to that from EST sequencing strategies.

A trial was established to assess the effects of harvesting dates and frequencies on the species, biomass and nutrient dynamics in wet grasslands in Belgium. Above and below-ground developments were analyzed. The vegetation changed evidently from the first spring after the establishment of the harvesting treatments onwards. One or two late summer and/or autumn harvests (August-October) led to lower productivity and higher species-richness. Generally, the reaction from the individual species was related to their capacity to germinate (Lychnis flos-cuculi L.) or initiate new tillers (Anthoxanthum odoratum L.), stolons (Agrostis canina L.) or rhizomes (Carex acuta L.). Not only did the total productivity decrease after one or two late summer and/or autumn harvests, but even the species increasing their cover/abundance after these treatments simultaneously developed less vigorous shoots. Soil analyses could not detect any changes in substrate fertility. However, analyses of nutrients in roots and rhizomes indicated lower concentrations of phosphorus and potassium in harvested conditions. Thus, the quick response of vegetation to the different treatments was related to its adapting species composition and to a depletion of phosphorus and potassium in roots and rhizomes. A depletion of nutrients in the soil was not detected.