Field and saccharification performances of poplars severely downregulated in CAD1.

Lignin is one of the main factors causing lignocellulosic biomass recalcitrance to enzymatic hydrolysis. Glasshouse-grown poplars severely downregulated for CINNAMYL ALCOHOL DEHYDROGENASE 1 (CAD1), the enzyme catalysing the last step in the monolignol-specific branch of lignin biosynthesis, have increased saccharification yields and normal growth. Here, we assess the performance of these hpCAD poplars in the field under short rotation coppice culture for two consecutive rotations of 1 yr and 3 yr. While 1-yr-old hpCAD wood had 10% less lignin, 3-yr-old hpCAD wood had wild-type lignin levels. Because of their altered cell wall composition, including elevated levels of cinnamaldehydes, both 1-yr-old and 3-yr-old hpCAD wood showed enhanced saccharification yields upon harsh alkaline pretreatments (up to +85% and +77%, respectively). In contrast with previous field trials with poplars less severely downregulated for CINNAMYL ALCOHOL DEHYDROGENASE (CAD), the hpCAD poplars displayed leaning phenotypes, early bud set, early flowering and yield penalties. Moreover, hpCAD wood had enlarged vessels, decreased wood density and reduced relative and free water contents. Our data show that the phenotypes of CAD-deficient poplars are strongly dependent on the environment and underpin the importance of field trials in translating basic research towards applications.

Canditate metabolites for ash dieback tolerance in Fraxinus excelsior.

Ash dieback, a forest epidemic caused by the invasive fungus Hymenoscyphus fraxineus, threatens ash trees throughout Europe. Within Fraxinus excelsior populations, a small proportion of genotypes show a low susceptibility to the pathogen. We compared the metabolomes from a cohort of low-susceptibility ash genotypes with a cohort of high-susceptibility ash genotypes. This revealed two significantly different chemotypes. A total of 64 candidate metabolites associated with reduced or increased susceptibility in the chemical families secoiridoids, coumarins, flavonoids, phenylethanoids, and lignans. Increased levels of two coumarins, fraxetin and esculetin, were strongly associated with reduced susceptibility to ash dieback. Both coumarins inhibited the growth of H. fraxineus in vitro when supplied at physiological concentrations, thereby validating their role as markers for low susceptibility to ash dieback. Similarly, fungal growth inhibition was observed when the methanolic bark extract of low-susceptibility ash genotypes was supplied. Our findings indicate the presence of constitutive chemical defense barriers against ash dieback in ash.

Transgenerational effects in asexually reproduced offspring of Populus.

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.

Variability in DNA Methylation and Generational Plasticity in the Lombardy Poplar, a Single Genotype Worldwide Distributed Since the Eighteenth Century.

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.

Variance, Genetic Control, and Spatial Phenotypic Plasticity of Morphological and Phenological Traits in Prunus spinosa and Its Large Fruited Forms (P. x fruticans).

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.

Adaptive mechanisms and genomic plasticity for drought tolerance identified in European black poplar (Populus nigra L.).

Summer droughts are likely to increase in frequency and intensity across Europe, yet long-lived trees may have a limited ability to tolerate drought. It is therefore critical that we improve our understanding of phenotypic plasticity to drought in natural populations for ecologically and economically important trees such as Populus nigra L. A common garden experiment was conducted using ∼500 wild P. nigra trees, collected from 11 river populations across Europe. Phenotypic variation was found across the collection, with southern genotypes from Spain and France characterized by small leaves and limited biomass production. To examine the relationship between phenotypic variation and drought tolerance, six genotypes with contrasting leaf morphologies were subjected to a water deficit experiment. 'North eastern' genotypes were collected at wet sites and responded to water deficit with reduced biomass growth, slow stomatal closure and reduced water use efficiency (WUE) assessed by Δ(13)C. In contrast, 'southern' genotypes originating from arid sites showed rapid stomatal closure, improved WUE and limited leaf loss. Transcriptome analyses of a genotype from Spain (Sp2, originating from an arid site) and another from northern Italy (Ita, originating from a wet site) revealed dramatic differences in gene expression response to water deficit. Transcripts controlling leaf development and stomatal patterning, including SPCH, ANT, ER, AS1, AS2, PHB, CLV1, ERL1-3 and TMM, were down-regulated in Ita but not in Sp2 in response to drought.

Repeated Summer Drought and Re-watering during the First Growing Year of Oak (Quercus petraea) Delay Autumn Senescence and Bud Burst in the Following Spring.

Climate change predicts harsher summer droughts for mid-latitudes in Europe. To enhance our understanding of the putative impacts on forest regeneration, we studied the response of oak seedlings (Quercus petraea) to water deficit. Potted seedlings originating from three locally sourced provenances were subjected to two successive drought periods during the first growing season each followed by a plentiful re-watering. Here, we describe survival and phenological responses after the second drought treatment, applying general linear mixed modeling. From the 441 drought treated seedlings 189 subsisted with higher chances of survival among smaller plants and among single plants per pot compared to doubles. Remarkably, survival was independent of the provenance, although relatively more plants had died off in two provenances compared to the third one with mean plant height being higher in one provenance and standard deviation of plant height being higher in the other. Timing of leaf senescence was clearly delayed after the severe drought treatment followed by re-watering, with two seedlings per pot showing a lesser retardation compared to single plants. This delay can be interpreted as a compensation time in which plants recover before entering the subsequent developmental process of leaf senescence, although it renders seedlings more vulnerable to early autumn frosts because of the delayed hardening of the shoots. Onset of bud flush in the subsequent spring still showed a significant but small delay in the drought treated group, independent of the number of seedlings per pot, and can be considered as an after effect of the delayed senescence. In both phenological models significant differences among the three provenances were detected independent from the treatment. The only provenance that is believed to be local of origin, displayed the earliest leaf senescence and the latest flushing, suggesting an adaptation to the local maritime climate. This provenance also displayed the highest standard deviation of plant height, which can be interpreted as an adaptation to variable and unpredictable weather conditions, favoring smaller plants in drought-prone summers and higher plants in more normal growing seasons.

Bud set in poplar--genetic dissection of a complex trait in natural and hybrid populations.

• The seasonal timing of growth events is crucial to tree distribution and conservation. The seasonal growth cycle is strongly adapted to the local climate that is changing because of global warming. We studied bud set as one cornerstone of the seasonal growth cycle in an integrative approach. • Bud set was dissected at the phenotypic level into several components, and phenotypic components with most genetic variation were identified. While phenotypic variation resided in the timing of growth cessation, and even so more in the duration from growth cessation to bud set, the timing of growth cessation had a stronger genetic component in both natural and hybrid populations. • Quantitative trait loci (QTL) were identified for the most discriminative phenotypic bud-set components across four poplar pedigrees. The QTL from different pedigrees were recurrently detected in six regions of the poplar genome. • These regions of 1.83-4.25 Mbp in size, containing between 202 and 394 genes, form the basis for further molecular-genetic dissection of bud set.

Genetical metabolomics of flavonoid biosynthesis in Populus: a case study.

Genetical metabolomics [metabolite profiling combined with quantitative trait locus (QTL) analysis] has been proposed as a new tool to identify loci that control metabolite abundances. This concept was evaluated in a case study with the model tree Populus. Using HPLC, the peak abundances were analyzed of 15 closely related flavonoids present in apical tissues of two full-sib poplar families, Populus deltoides cv. S9-2 x P. nigra cv. Ghoy and P. deltoides cv. S9-2 x P. trichocarpa cv. V24, and correlation and QTL analysis were used to detect flux control points in flavonoid biosynthesis. Four robust metabolite quantitative trait loci (mQTL), associated with rate-limiting steps in flavonoid biosynthesis, were mapped. Each mQTL was involved in the flux control to one or two flavonoids. Based on the identities of the affected metabolites and the flavonoid pathway structure, a tentative function was assigned to three of these mQTL, and the corresponding candidate genes were mapped. The data indicate that the combination of metabolite profiling with QTL analysis is a valuable tool to identify control points in a complex metabolic pathway of closely related compounds.