Perspectives on Populus spp. (Salicaceae) bud extracts as antioxidant and anti-inflammatory agents.

Hydroalcoholic extracts obtained from buds of P. nigra, P. deltoides and P. trichocarpa were characterized by HPLC-DAD-MS analysis and subsequently evaluated in vitro for their antioxidant and anti-inflammatory activities. ABTS and DPPH assays evidenced that P. nigra showed the best antioxidant activity in line with its highest total phenolic content. The analysis of the anti-inflammatory activity clearly demonstrated that all extracts suppressed the production of key pro-inflammatory cytokines (IL-6, Il-1ß and TNF-α) and HMGB1 inflammatory danger signal. These results show antioxidant and critical anti-inflammatory activities mediated by the extracts, emphasising their potentiality as therapeutic agents.

A single gene underlies the dynamic evolution of poplar sex determination.

Although hundreds of plant lineages have independently evolved dioecy (that is, separation of the sexes), the underlying genetic basis remains largely elusive1. Here we show that diverse poplar species carry partial duplicates of the ARABIDOPSIS RESPONSE REGULATOR 17 (ARR17) orthologue in the male-specific region of the Y chromosome. These duplicates give rise to small RNAs apparently causing male-specific DNA methylation and silencing of the ARR17 gene. CRISPR-Cas9-induced mutations demonstrate that ARR17 functions as a sex switch, triggering female development when on and male development when off. Despite repeated turnover events, including a transition from the XY system to a ZW system, the sex-specific regulation of ARR17 is conserved across the poplar genus and probably beyond. Our data reveal how a single-gene-based mechanism of dioecy can enable highly dynamic sex-linked regions and contribute to maintaining recombination and integrity of sex chromosomes.

Correction to: Long-term study of a subdioecious Populus ×canescens family reveals sex lability of females and reproduction behaviour of cosexual plants.

Table 4 in the original publication reports incomplete genotype names in the column "Cross" and wrong codes in the column "Generation".

Long-term study of a subdioecious Populus ×canescens family reveals sex lability of females and reproduction behaviour of cosexual plants.

KEY MESSAGE: Cosexual Populus ×canescens plants are inconstant females with life course plasticity of sex phenotype and can reproduce by selfing. Populus species are dioecious, but deviations from dioecy are reported in some cases. The objectives of this study were to investigate the phenotypic expression and the inheritance of subdioecy in a Populus ×canescens pedigree. The F1 progeny was monitored for sex during 14 years. Thirty per cent of individuals expressed deviations from dioecy and long-term plasticity of sex. Some plants started flowering as male, then became cosexual, and finally turned female. Two cosexual individuals were self-pollinated and generated a selfed progeny markedly impaired by inbreeding depression, but able to reproduce by outcrossing. Sex segregation of the F1 progeny statistically fitted the expected ratio 1:2:1 (female:male:cosexual). By analysis of DNA markers, the cosexual individuals were genetically clustered with the females. The segregation ratio and the genetic profile indicated that cosexual plants were female with altered sex phenotype. Linkage analysis identified a putative sex-determining region with suppressed recombination on chromosome 19 of the male Populus tremula parent. The male sex trait was linked to the pericentromeric region of the P. tremula chromosome 19, whereas the cosexual trait was linked to chromosome 19 of the female Populus alba parent. A genetic model is proposed to explain inheritance and phenotypic expression of sex.

Genetic variation for leaf morphology, leaf structure and leaf carbon isotope discrimination in European populations of black poplar (Populus nigra L.).

To buffer against the high spatial and temporal heterogeneity of the riparian habitat, riparian tree species, such as black poplar (Populus nigra L.), may display a high level of genetic variation and phenotypic plasticity for functional traits. Using a multisite common garden experiment, we estimated the relative contribution of genetic and environmental effects on the phenotypic variation expressed for individual leaf area, leaf shape, leaf structure and leaf carbon isotope discrimination (Δ(13)C) in natural populations of black poplar. Twenty-four to 62 genotypes were sampled in nine metapopulations covering a latitudinal range from 48 °N to 42 °N in France and in Italy and grown in two common gardens at Orléans (ORL) and at Savigliano (SAV). In the two common gardens, substantial genetic variation was expressed for leaf traits within all metapopulations, but its expression was modulated by the environment, as attested by the genotype × environment (G × E) interaction variance being comparable to or even greater than genetic effects. For LA, G × E interactions were explained by both changes in genotype ranking between common gardens and increased variation in SAV, while these interactions were mainly attributed to changes in genotype ranking for Δ(13)C. The nine P. nigra metapopulations were highly differentiated for LA, as attested by the high coefficient of genetic differentiation (QST = 0.50 at ORL and 0.51 at SAV), and the pattern of metapopulation differentiation was highly conserved between the two common gardens. In contrast, they were moderately differentiated for Δ(13)C (QST = 0.24 at ORL and 0.25 at SAV) and the metapopulation clustering changed significantly between common gardens. Our results evidenced that the nine P. nigra metapopulations present substantial genetic variation and phenotypic plasticity for leaf traits, which both represent potentially significant determinants of populations' capacities to respond, on a short-term basis and over generations, to environmental variations.

Phenotypic plasticity, QTL mapping and genomic characterization of bud set in black poplar.

BACKGROUND: The genetic control of important adaptive traits, such as bud set, is still poorly understood in most forest trees species. Poplar is an ideal model tree to study bud set because of its indeterminate shoot growth. Thus, a full-sib family derived from an intraspecific cross of P. nigra with 162 clonally replicated progeny was used to assess the phenotypic plasticity and genetic variation of bud set in two sites of contrasting environmental conditions. RESULTS: Six crucial phenological stages of bud set were scored. Night length appeared to be the most important signal triggering the onset of growth cessation. Nevertheless, the effect of other environmental factors, such as temperature, increased during the process. Moreover, a considerable role of genotype × environment (G × E) interaction was found in all phenological stages with the lowest temperature appearing to influence the sensitivity of the most plastic genotypes.Descriptors of growth cessation and bud onset explained the largest part of phenotypic variation of the entire process. Quantitative trait loci (QTL) for these traits were detected. For the four selected traits (the onset of growth cessation (date2.5), the transition from shoot to bud (date1.5), the duration of bud formation (subproc1) and bud maturation (subproc2)) eight and sixteen QTL were mapped on the maternal and paternal map, respectively. The identified QTL, each one characterized by small or modest effect, highlighted the complex nature of traits involved in bud set process. Comparison between map location of QTL and P. trichocarpa genome sequence allowed the identification of 13 gene models, 67 bud set-related expressional and six functional candidate genes (CGs). These CGs are functionally related to relevant biological processes, environmental sensing, signaling, and cell growth and development. Some strong QTL had no obvious CGs, and hold great promise to identify unknown genes that affect bud set. CONCLUSIONS: This study provides a better understanding of the physiological and genetic dissection of bud set in poplar. The putative QTL identified will be tested for associations in P. nigra natural populations. The identified QTL and CGs will also serve as useful targets for poplar breeding.

Accelerating the domestication of forest trees in a changing world.

In light of impending water and arable land shortages, population growth and climate change, it is more important than ever to examine how forest tree domestication can be accelerated to sustainably meet future demands for wood, biomass, paper, fuel and biomaterials. Because of long breeding cycles, tree domestication cannot be rapidly achieved through traditional genetic improvement methods alone. Integrating modern genetic and genomic techniques with conventional breeding will expedite tree domestication. Breeders will only embrace these technologies if they are cost-effective and readily accessible, and forest landowners will only adopt end-products that meet with regulatory approval and public acceptance. All parties involved must work together to achieve these objectives for the benefit of society.

Intraspecific variation of physiological and molecular response to cadmium stress in Populus nigra L.

Little is known about the variability of response to heavy metal stress within tree species, although it could be a key for a better understanding of tolerance mechanisms and for breeding. The aim of the present study was to characterize the natural variation of response to cadmium (Cd) in Populus nigra L. in order to understand the mechanisms of Cd tolerance. For that, two P. nigra genotypes, originating from contrasting environments in northern (genotype 58-861) and southern (genotype Poli) Italy, were exposed to Cd stress in hydroponics for 3 weeks. The effect of stress was estimated by measuring biomass production, photosynthetic performance and accumulation and translocation of Cd at the end of the experiment. To better understand the mechanisms of Cd tolerance, the expression of some candidate genes involved in the ascorbate-glutathione cycle (ascorbate peroxidase, glutathione reductase, glutathione S-transferase) and in metal sequestration (metallothioneins) was analyzed in leaves. Biomass production and photosynthesis were affected by the treatment in both clones but the southern clone was markedly more tolerant to Cd stress than the other. Nevertheless, the Cd content in leaves was not significantly different between the two clones and was quite low compared to other species. The content of thiols and phytochelatins (PCs), associated with the transcription profile of the glutathione S-transferase gene, indicated relevant differences in the use of the PCs pathway under Cd stress, which could explain the different tolerance to Cd. The northern clone accumulated thiols but down-regulated the GST gene, whereas the southern clone accumulated PCs and up-regulated the GST gene, which can be useful to complex and detoxify Cd. These results suggest that the glutathione pathway is involved in the differential Cd tolerance of the two genotypes. The natural germplasm of P. nigra represents a valuable resource for understanding tolerance to Cd and for selection of plant material for phytoremediation.

Comparative study of transcriptional and physiological responses to salinity stress in two contrasting Populus alba L. genotypes.

Soil salinity is an important limiting factor to tree growth and productivity. Populus alba L. is a moderately salt-tolerant species and its natural populations are adapted to contrasting environments, thus providing genetic resources to identify key genes for tolerance to abiotic stress, such as salinity. To elucidate the molecular and genetic basis of variation for salinity tolerance in P. alba, we analyzed the short-term ecophysiological and transcriptome response to salinity. Two contrasting genotypes, 6K3, salt sensitive, and 14P11, salt tolerant, originating from North and South Italy, respectively, were challenged with salt stress (200 mM NaCl). Sodium accumulated in the leaves of salt-treated plants and its concentration increased with time. The net photosynthesis was strongly reduced by salinity in both genotypes, with 6K3 being significantly more affected than 14P11. The transcriptional changes in leaves were analyzed using cDNA microarrays containing about 7000 stress-related poplar expressed sequence tags (EST). A microarray experiment based on RNA pooling showed a number of salinity--regulated transcripts that markedly increased from 3 h to 3 days of salinity treatment. Thus, a detailed analysis was performed on replicated plants collected at 3 days, when ~20% of transcripts showed significant change induced by salinity. In 6K3, there were more genes with decreased expression than genes with increased expression, whereas such a difference was not found in 14P11. Most transcripts with decreased expression were shared between the two genotypes, whereas transcripts with increased expression were mostly regulated in a genotype-specific manner. The commonly decreased transcripts (71 genes) were functionally related to carbohydrate metabolism, energy metabolism and photosynthesis. These biological processes were consistent with the strong inhibition of photosynthesis, caused by salinity. The commonly increased transcripts (13 genes) were functionally related to primary metabolism and biosynthesis of proteins and macromolecules. The salinity-increased transcripts discriminated the molecular response of the two genotypes. In 14P11, the 21 genes specifically salinity-induced were related to stress response, cell development, cell death and catabolism. In 6K3, the 15 genes with salinity-increased expression were involved in protein biosynthesis, metabolism of macromolecules and cell organization and biogenesis. The difference in transcriptome response between the two genotypes could address the molecular basis of intra-specific variation of salinity tolerance in P. alba.

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.

Development of a novel set of EST-SSR markers and cross-species amplification in Tamarix africana (Tamaricaceae).

PREMISE OF THE STUDY: Tamarix plants are resistant to abiotic stresses and have become invasive in North America. Their taxonomy is troublesome, and few molecular makers are available to enable species identification or to track the spread of specific invasive genotypes. Transcriptome sequencing projects offer a potential source for the development of new markers. • METHODS AND RESULTS: Thirteen polymorphic simple sequence repeats (SSRs) markers derived from Expressed Sequence Tags (ESTs) from Tamarix hispida, T. androssowii, T. ramosissima, and T. albiflonum were identified and screened on 24 samples of T. africana to detect polymorphism. The number of alleles per locus ranged from two to eight, with an average of 4.3 alleles per locus, and the mean expected heterozygosity was 0.453. • CONCLUSIONS: Amplification products of these 13 loci were also generated for T. gallica. These new EST-SSR markers will be useful in genetic characterization of Tamarix, as additional tools for taxonomic clarification, and for studying invasive populations where they are a threat.

Allele-specific PCR in SNP genotyping.

The increasing need for large-scale genotyping applications of single nucleotide polymorphisms (SNPs) in model and nonmodel organisms requires the development of low-cost technologies accessible to minimally equipped laboratories. The method presented here allows efficient discrimination of SNPs by allele-specific PCR in a single reaction with standard PCR conditions. A common reverse primer and two forward allele-specific primers with different tails amplify two allele-specific PCR products of different lengths, which are further separated by agarose gel electrophoresis. PCR specificity is improved by the introduction of a destabilizing mismatch within the 30 end of the allele-specific primers. This is a simple and inexpensive method for SNP detection that does not require PCR optimization.

Relationships among productivity determinants in two hybrid poplar families grown during three years at two contrasting sites.

The objective of this study was to evaluate the environmental, temporal and genetic stability of the relationships between growth and a selection of tree architectural, leaf and phenological traits (selection based on the conclusions of previous studies carried out on the same experimental trial). Therefore, the growth of two hybrid families, Populus deltoides 'S9-2' x Populus nigra 'Ghoy' (D x N family, 180 F(1)) and P. deltoides 'S9-2' x Populus trichocarpa 'V24' (D x T family, 182 F(1)), was investigated during a 3-year period at two sites, i.e., in northern Italy and central France. At the end of the second growing season, all trees were coppiced and the resprouts were thinned to a single stem. At the end of each growing season, stem circumference and height were measured for all F(1) hybrids. The number of sylleptic branches, individual leaf area (LA) and petiole length of the largest leaf along the main stem, production of new leaves, bud flush and bud set were estimated for a selection of genotypes (31 F(1)) per family at each site during the course of the 3-year experiment. The D x T family was clearly the most productive family and displayed the highest heterosis values. However, there appeared to be a compromise between good growth at a given site and stability between the two different sites, both at family and at genotype levels. Particularly, the less performing trees were stable between Italy and France. Among the studied growth components, the number of sylleptic branches and individual LA of the largest leaf along the main stem were the best growth predictors, irrespective of site and family. Growth strategies in terms of leaf development differed between the two families. Hence, leaf production rate was strongly associated with growth of the D x N family only. These results have important consequences for the use of the studied traits as selection criteria in breeding programmes.

Plasticity of growth and sylleptic branchiness in two poplar families grown at three sites across Europe.

Two hybrid poplar maternal half-sib families, resulting from controlled crosses of the female parent Populus deltoides 'S9-2' with P. nigra 'Ghoy' and P. trichocarpa 'V24', were grown at three sites: northern Italy, central France and southern England. Juvenile stem growth traits (height, circumference and volume) and sylleptic branchiness (number of branches, density of branches per unit of stem height, percentage of the stem carrying branches and distance of the highest sylleptic branch to the top of the stem) were measured on 1-year-old shoots. Our general objectives were to determine the degree to which the expression of stem growth and syllepsis and the relationships between them are affected by environmental conditions and to evaluate the efficiency of indirect selection for stem growth using branching traits as secondary criteria. The performance of both families differed significantly within and between sites. Pronounced heterosis was observed and highly significant genotype x environment interactions were found for all traits across the sites. Syllepsis showed more marked genetic variation and plasticity than stem growth traits. Relationships between sylleptic branchiness and stem growth depended on environmental conditions. Heritability values at the individual level ranged between 0.09 and 0.59, but genetic gain in stem volume was not significantly improved when selection was based on sylleptic branch characteristics. However, despite strong phenotypic plasticity among sites, genotypic ranking among the sites was relatively stable.

Stomatal conductance and not stomatal density determines the long-term reduction in leaf transpiration of poplar in elevated CO2.

Using a free-air CO2 enrichment (FACE) experiment, poplar trees (Populus x euramericana clone I214) were exposed to either ambient or elevated [CO2] from planting, for a 5-year period during canopy development, closure, coppice and re-growth. In each year, measurements were taken of stomatal density (SD, number mm(-2)) and stomatal index (SI, the proportion of epidermal cells forming stomata). In year 5, measurements were also taken of leaf stomatal conductance (gs, micromol m(-2) s(-1)), photosynthetic CO2 fixation (A, mmol m(-2) s(-1)), instantaneous water-use efficiency (A/E) and the ratio of intercellular to atmospheric CO2 (Ci:Ca). Elevated [CO2] caused reductions in SI in the first year, and in SD in the first 2 years, when the canopy was largely open. In following years, when the canopy had closed, elevated [CO2] had no detectable effects on stomatal numbers or index. In contrast, even after 5 years of exposure to elevated [CO2], gs was reduced, A/E was stimulated, and Ci:Ca was reduced relative to ambient [CO2]. These outcomes from the long-term realistic field conditions of this forest FACE experiment suggest that stomatal numbers (SD and SI) had no role in determining the improved instantaneous leaf-level efficiency of water use under elevated [CO2]. We propose that altered cuticular development during canopy closure may partially explain the changing response of stomata to elevated [CO2], although the mechanism for this remains obscure.

Do above-ground growth dynamics of poplar change with time under CO2 enrichment?

• In a free-air CO2 enrichment (FACE) study, above-ground growth of Populus alba, Populus nigra and Populus×euramericana was followed continuously during the first rotation cycle of a short rotation culture (SRC) plantation to test possible changes in the response to elevated CO2 occurring from planting until canopy closure. • Height, stem basal area, stem volume index, branch production, and bud phenology were monitored for 3 yr. Moreover the coefficient of variation and a competition index were calculated to analyse the onset and the typology of competition. • Volume index was higher under elevated CO2 by 77%, 24% and 22%, as mean value for the three species, in the first, second and third years, respectively. The stimulating response, although univocal, differed in extent among species. Branch production was stimulated only in the first year, whereas bud phenology was unaffected. • The analysis of these results show that growth was stimulated by elevated CO2 only in the first year, although differences in volume index remained significant even in the second and third years. In the third year, under canopy closure, only competitively advantaged individuals profited by the FACE treatment.