Genome-Wide Transcriptomic and Metabolomic Analyses Unveiling the Defence Mechanisms of Populus tremula against Sucking and Chewing Insect Herbivores.

Plants and insects coevolved as an evolutionarily successful and enduring association. The molecular arms race led to evolutionary novelties regarding unique mechanisms of defence and detoxification in plants and insects. While insects adopt mechanisms to conquer host defence, trees develop well-orchestrated and species-specific defence strategies against insect herbivory. However, current knowledge on the molecular underpinnings of fine-tuned tree defence responses against different herbivore insects is still restricted. In the current study, using a multi-omics approach, we unveiled the defence response of Populus tremula against aphids (Chaitophorus populialbae) and spongy moths (Lymantria dispar) herbivory. Comparative differential gene expression (DGE) analyses revealed that around 272 and 1203 transcripts were differentially regulated in P. tremula after moth and aphid herbivory compared to uninfested controls. Interestingly, 5716 transcripts were differentially regulated in P. tremula between aphids and moth infestation. Further investigation showed that defence-related stress hormones and their lipid precursors, transcription factors, and signalling molecules were over-expressed, whereas the growth-related counterparts were suppressed in P. tremula after aphid and moth herbivory. Metabolomics analysis documented that around 37% of all significantly abundant metabolites were associated with biochemical pathways related to tree growth and defence. However, the metabolic profiles of aphid and moth-fed trees were quite distinct, indicating species-specific response optimization. After identifying the suitable reference genes in P. tremula, the omics data were further validated using RT-qPCR. Nevertheless, our findings documented species-specific fine-tuning of the defence response of P. tremula, showing conservation on resource allocation for defence overgrowth under aphid and moth herbivory. Such findings can be exploited to enhance our current understanding of molecular orchestration of tree responses against herbivory and aid in developing insect pest resistance P. tremula varieties.

The effect of nitrogen source and levels on hybrid aspen tree physiology and wood formation.

Nitrogen can be taken up by trees in the form of nitrate, ammonium and amino acids, but the influence of the different forms on tree growth and development is poorly understood in angiosperm species like Populus. We studied the effects of both organic and inorganic forms of nitrogen on growth and wood formation of hybrid aspen trees in experimental conditions that allowed growth under four distinct steady-state nitrogen levels. Increased nitrogen availability had a positive influence on biomass accumulation and the radial dimensions of both xylem vessels and fibers, and a negative influence on wood density. An optimal level of nitrogen availability was identified where increases in biomass accumulation outweighed decreases in wood density. None of these responses depended on the source of nitrogen except for shoot biomass accumulation, which was stimulated more by treatments complemented with nitrate than by ammonium alone or the organic source arginine. The most striking difference between the nitrogen sources was the effect on lignin composition, whereby the abundance of H-type lignin increased only in the presence of nitrate. The differential effect of nitrate is possibly related to the well-known role of nitrate as a signaling compound. RNA-sequencing revealed that while the lignin-biosynthetic genes did not significantly (FDR <0.01) respond to added NO3 - , the expression of several laccases, catalysing lignin polymerization, was dependent on N-availability. These results reveal a unique role of nitrate in wood formation and contribute to the knowledge basis for decision-making in utilizing hybrid aspen as a bioresource.

Seasonal dynamics of cell-to-cell transport in angiosperm wood.

This study describes the seasonal changes in cell-to-cell transport in three selected angiosperm tree species, Acer pseudoplatanus (maple), Fraxinus excelsior (ash), and Populus tremula × tremuloides (poplar), with an emphasis on the living wood component, xylem parenchyma cells (XPCs). We performed anatomical studies, dye loading through the vascular system, measurements of non-structural carbohydrate content, immunocytochemistry, inhibitory assays and quantitative real-time PCR to analyse the transport mechanisms and seasonal variations in wood. The abundance of membrane dye in wood varied seasonally along with seasonally changing tree phenology, cambial activity, and non-structural carbohydrate content. Moreover, dyes internalized in vessel-associated cells and 'trapped' in the endomembrane system are transported farther between other XPCs via plasmodesmata. Finally, various transport mechanisms based on clathrin-mediated and clathrin-independent endocytosis, and membrane transporters, operate in wood, and their involvement is species and/or season dependent. Our study highlights the importance of XPCs in seasonally changing cell-to-cell transport in both ring-porous (ash) and diffuse-porous (maple, poplar) tree species, and demonstrates the involvement of both endocytosis and plasmodesmata in intercellular communication in angiosperm wood.

Flexure wood formation via growth reprogramming in hybrid aspen involves jasmonates and polyamines and transcriptional changes resembling tension wood development.

Stem bending in trees induces flexure wood but its properties and development are poorly understood. Here, we investigated the effects of low-intensity multidirectional stem flexing on growth and wood properties of hybrid aspen, and on its transcriptomic and hormonal responses. Glasshouse-grown trees were either kept stationary or subjected to several daily shakes for 5 wk, after which the transcriptomes and hormones were analyzed in the cambial region and developing wood tissues, and the wood properties were analyzed by physical, chemical and microscopy techniques. Shaking increased primary and secondary growth and altered wood differentiation by stimulating gelatinous-fiber formation, reducing secondary wall thickness, changing matrix polysaccharides and increasing cellulose, G- and H-lignin contents, cell wall porosity and saccharification yields. Wood-forming tissues exhibited elevated jasmonate, polyamine, ethylene and brassinosteroids and reduced abscisic acid and gibberellin signaling. Transcriptional responses resembled those during tension wood formation but not opposite wood formation and revealed several thigmomorphogenesis-related genes as well as novel gene networks including FLA and XTH genes encoding plasma membrane-bound proteins. Low-intensity stem flexing stimulates growth and induces wood having improved biorefinery properties through molecular and hormonal pathways similar to thigmomorphogenesis in herbaceous plants and largely overlapping with the tension wood program of hardwoods.

Impact of xylan on field productivity and wood saccharification properties in aspen.

Xylan that comprises roughly 25% of hardwood biomass is undesirable in biorefinery applications involving saccharification and fermentation. Efforts to reduce xylan levels have therefore been made in many species, usually resulting in improved saccharification. However, such modified plants have not yet been tested under field conditions. Here we evaluate the field performance of transgenic hybrid aspen lines with reduced xylan levels and assess their usefulness as short-rotation feedstocks for biorefineries. Three types of transgenic lines were tested in four-year field tests with RNAi constructs targeting either Populus GT43 clades B and C (GT43BC) corresponding to Arabidopsis clades IRX9 and IRX14, respectively, involved in xylan backbone biosynthesis, GATL1.1 corresponding to AtGALT1 involved in xylan reducing end sequence biosynthesis, or ASPR1 encoding an atypical aspartate protease. Their productivity, wood quality traits, and saccharification efficiency were analyzed. The only lines differing significantly from the wild type with respect to growth and biotic stress resistance were the ASPR1 lines, whose stems were roughly 10% shorter and narrower and leaves showed increased arthropod damage. GT43BC lines exhibited no growth advantage in the field despite their superior growth in greenhouse experiments. Wood from the ASPR1 and GT43BC lines had slightly reduced density due to thinner cell walls and, in the case of ASPR1, larger cell diameters. The xylan was less extractable by alkali but more hydrolysable by acid, had increased glucuronosylation, and its content was reduced in all three types of transgenic lines. The hemicellulose size distribution in the GALT1.1 and ASPR1 lines was skewed towards higher molecular mass compared to the wild type. These results provide experimental evidence that GATL1.1 functions in xylan biosynthesis and suggest that ASPR1 may regulate this process. In saccharification without pretreatment, lines of all three constructs provided 8-11% higher average glucose yields than wild-type plants. In saccharification with acid pretreatment, the GT43BC construct provided a 10% yield increase on average. The best transgenic lines of each construct are thus predicted to modestly outperform the wild type in terms of glucose yields per hectare. The field evaluation of transgenic xylan-reduced aspen represents an important step towards more productive feedstocks for biorefineries.

Molecular studies of rust on European aspen suggest an autochthonous relationship shaped by genotype.

Forests are at increasing risk from pathogen outbreak. Climate change for example enhance the risk of local disease outbreaks, and naturalization of exotic pathogens may follow human activities, warranting robust pest surveillance routines to support forest management. Melampsora pinitorqua (pine twisting rust) is of concern in Swedish forestry, and here we evaluate the use of visible rust scores (VRS) on its obligate summer host, European aspen (Populus tremula) as a tool for quantification of the pathogen. With use of species-specific primers, we could detect the native rust, but we failed to detect two exotic rusts (M. medusae and M. larici-populina). We found that aspen genotype determined the presence of fungal genetic markers (amplifying the ITS2 region of the fungal rDNA sequence) as well as DNA sequences specific to M. pinitorqua. We correlated VRS with the amount of fungal DNA in the same leaf, and we related the findings to aspen genotype-specific parameters such as the ability to synthesize and store leaf condensed tannins (CT). At the genotype level both positive and negative relationships were observed between CTs, fungal markers, and rust infestations. However, at the population level, foliar CT concentrations correlated negatively with general fungal- and rust-specific marker abundances. Our results, therefore, do not support the use of VRS to assess Melampsora infestation in Aspen. They do, however, suggest that the relationship between European aspen and rust infestation may be characterized as autochthonous in northern Sweden.

Preparation of alkali lignin extracted from ligno-cellulosic populus tremula fibers: Application to copper oxide nanoparticles synthesis, characterization, and methylene blue biosorption study.

The green synthesis of nanoparticles using biogenic approaches constitutes a challenge for effective applications. The massive aliphatic hydroxyl groups of lignin exhibited excellent reduction properties allowing the production of metallic nanoparticles. In this work, alkali lignin was extracted from virgin populus tremula and used for the preparation of copper oxide nanoparticles. The analysis of the prepared nanoparticles was assessed using Fourier Transform Infra-red (FT-IR), X-Ray Diffraction (XRD), Scanning Electron Microscopy (SEM), Energy Dispersive X-ray spectroscopy (EDX), and Transmission Electron Microscopy (TEM). FT-IR results displayed that different phytochemicals constituents of lignin extract were responsible for the production of CuO nanoparticles. XRD information demonstrated monoclinic CuO nanoparticles with a mean size of 12.4 nm. SEM images showed that some nanoparticles were quite separated from each other and some of them were agglomerated due to the oxidation of metal nanoparticles. TEM photos indicated that the overlap of the nanoparticles resulted in rectangular patterns due to the presence of lignin on the surface of CuO nanoparticles. Finally, the prepared CuO nanoparticles were applied for the removal of methylene blue from water. The results showed that the maximum adsorption capacity reached 85 mg/g at the following conditions: T = 20 °C, pH = 6, and time = 60 min.

Influence of Tree Species and Size on Bark Browsing by Large Wild Herbivores.

Although an important part of the ecosystem, large wild herbivores (LWH), especially red deer (Cervus elaphus L.), cause significant damage to economically valuable timber in forests of Central Europe. Recent work has demonstrated that less valuable softwood broadleaved trees can act as "biological control" that helps reduce bark browsing on more valuable trees in a mixed stand. To better understand the factors that influence how much bark area and mass are removed by LWH from these broadleaved trees, we took advantage of a novel "natural" experiment that occurred after a breach in a herbivory exclosure surrounding a 10-year old mixed broadleaved/conifer stand in the Western Carpathians in north-western Slovakia. We measured the area of old (up to 2 years previously) and new browsed patches on stems of common aspen (Populus tremula L.), common rowan (Sorbus aucuparia L.) and goat willow (Salix caprea L.), and their position along the vertical profile of the stem. The browsed bark area (cm2) was then converted to the bark mass (g) removed and the proportion of browsed bark to total bark (%) using conversion equations. Our models demonstrated that the amount of bark removed was influenced by tree species, stem diameter, age of browsing (old vs. new), and stem section along the vertical profile. LWH removed the most bark area from willow but the most bark mass from aspen because aspen had thicker bark than the other tree species. Bark browsing was greater on trees > 6 cm basal diameter. The distribution of bark browsing along the vertical profile was symmetrical (unimodal) with maximum intensity at 101−125 cm from the ground, which corresponds with the height most optimal for feeding by red deer. However, previous browsing in 2019 and 2020 caused new browsing on willow in 2021 to be focused in stem sections lower (51−75 cm) and higher (126−150 cm) than that optima. By quantifying browsing patterns and the amount of bark that is accessible to LWH for forage on the most attractive softwood broadleaved trees, our work will contribute to developing better methods for protecting commercially important species such as European beech (Fagus sylvatica L.) and Norway spruce (Picea abies L. Karst.) in areas of Central Europe that are greatly affected by increasing population density of LWH, especially red deer.

Effects of condensed tannins on behavior and performance of a specialist aphid on aspen.

Genes involved in plant defences against herbivores and pathogens are often highly polymorphic. This is a putative sign that balancing selection may have operated reciprocally on the hosts and their herbivores. Spatial and temporal variations (for example, in soil nutrients and the plants' ontogenetic development) may also modulate resistance traits, and thus selection pressures, but have been largely overlooked in theories of plant defences. Important elements of defences in Populus tremula (hereafter aspen) are phenolic compounds, including condensed tannins (CTs). Concentrations of CTs vary considerably with both variations in external factors and time, but they are also believed to provide genotype-dependent resistance, mainly against chewing herbivores and pathogens. However, evidence of their contributions to resistance is sparse. Detailed studies of co-evolved plant-herbivore associations could provide valuable insights into these contributions. Therefore, we examined correlations between CT levels in aspen leaves and both the feeding behavior and reproduction of the specialist aspen leaf aphid (Chaitophorus tremulae) in varied conditions. We found that xylem sap intake and probing difficulties were higher on genotypes with high-CT concentrations. However, aphids engaged in more nonprobing activities on low-CT genotypes, indicating that CTs were not the only defence traits involved. Thus, high-CT genotypes were not necessarily more resistant than low-CT genotypes, but aphid reproduction was generally negatively correlated with local CT accumulation. Genotype-specific resistance ranking also depended on the experimental conditions. These results support the hypothesis that growth conditions may affect selection pressures mediated by aphids in accordance with balancing selection theory.

Aspen Leaves as a "Chemical Landscape" for Fungal Endophyte Diversity-Effects of Nitrogen Addition.

Abiotic and biotic factors may shape the mycobiome communities in plants directly but also indirectly by modifying the quality of host plants as a substrate. We hypothesized that nitrogen fertilization (N) would determine the quality of aspen (Populus tremula) leaves as a substrate for the endophytic fungi, and that by subjecting the plants to N, we could manipulate the concentrations of positive (nutritious) and negative (antifungal) chemicals in leaves, thus changing the internal "chemical landscape" for the fungi. We expected that this would lead to changes in the fungal community composition, in line with the predictions of heterogeneity-diversity relationship and resource availability hypotheses. To test this, we conducted a greenhouse study where aspen plants were subjected to N treatment. The chemical status of the leaves was confirmed using GC/MS (114 metabolites, including amino acids and sugars), LC/MS (11 phenolics), and UV-spectrometry (antifungal condensed tannins, CTs), and the endophytic communities were characterized using culture-dependent sequencing. We found that N treatment reduced foliar concentrations of CT precursor catechin but not that of CTs. Nitrogen treatment also increased the concentrations of the amino acids and reduced the concentration of some sugars. We introduced beetle herbivores (H) as a second treatment but found no rapid changes in chemical traits nor strong effect on the diversity of endophytes induced by herbivores. A few rare fungi were associated with and potentially vectored by the beetle herbivores. Our findings indicate that in a controlled environment, the externally induced changes did not strongly alter endophyte diversity in aspen leaves.

Extraction of Cellulose Polymeric Material from Populus tremula Fibers: Characterization and Application to the Adsorption of Methylene Blue and Crystal Violet.

Cellulose is the most widely available biopolymer which is extensively used for several applications including textiles, composites, pharmaceutical, water treatment, etc. In this investigation, cellulose was chemically extracted from Populus tremula seed fibers. Samples were characterized using FT-IR, SEM, XRD, and TGA-DTA analyses. FT-IR spectrum of the extracted cellulose confirmed that hemicellulose and lignin were removed during alkali and bleaching treatments. SEM images showed the partially roughened surface of the fiber due to the removal of non-cellulosic elements and surface impurities during chemical modification. The crystallinity index values for untreated Populus tremula fibers and extracted cellulose were calculated to be 32.8% and 58.9%, respectively. The obvious increase in the crystallinity index for the extracted cellulose confirmed the removal of amorphous compounds present in raw populus. Alkali-treated populus fibers were more thermally stable than raw fibers. All changes observed after alkali and bleaching treatments evidenced the removal of amorphous contents and non-cellulosic components in raw populus fibers. Extracted cellulose exhibited excellent adsorption capacities of methylene blue (140.4 mg g-1) and crystal violet (154 mg g-1). The pseudo second order equation fitted well the kinetic data indicating a chemi-sorption process. The Freundlich model complied well with the experimental data suggesting that the adsorption of the studied dyes was multilayer.

Impact of Plant-Associated Bacteria on the In Vitro Growth and Pathogenic Resistance against Phellinus tremulae of Different Aspen (Populus) Genotypes.

Aspens (Populus tremula and its hybrids), economically and ecologically important fast-growing trees, are often damaged by Phellinus tremulae, a rot-causing fungus. Plant-associated bacteria can be used to increase plant growth and resistance; however, no systematic studies relating the activity of symbiotic bacteria to aspen resistance against Phellinus tremulae have been conducted so far. The present pioneer study investigated the responses of two Populus tremula and two P. tremula × P. tremuloides genotypes to in vitro inoculations with, first, either Pseudomonas sp. or Paenibacillus sp. bacteria (isolated originally from hybrid aspen tissue cultures and being most closely related to Pseudomonas oryzihabitans and Paenibacillus tundrae, respectively) and, in the subsequent stage, with Phellinus tremulae. Both morphological parameters of in vitro-grown plants and biochemical content of their leaves, including photosynthesis pigments and secondary metabolites, were analyzed. It was found that both Populus tremula × P. tremuloides genotypes, whose development in vitro was significantly damaged by Phellinus tremulae, were characterized by certain responses to the studied bacteria: decreased shoot development by both Paenibacillus sp. and Pseudomonas sp. and increased phenol content by Pseudomonas sp. In turn, these responses were lacking in both Populus tremula genotypes that showed in vitro resistance to the fungus. Moreover, these genotypes showed positive long-term growth responses to bacterial inoculation, even synergistic with the subsequent fungal inoculation. Hence, the studied bacteria were demonstrated as a potential tool for the improved in vitro propagation of fungus-resistant aspen genotypes.

Saccharification Potential of Transgenic Greenhouse- and Field-Grown Aspen Engineered for Reduced Xylan Acetylation.

High acetylation of xylan in hardwoods decreases their value as biorefinery feedstocks. To counter this problem, we have constitutively suppressed RWA genes encoding acetyl-CoA transporters using the 35S promoter, or constitutively and wood-specifically (using the WP promoter) expressed fungal acetyl xylan esterases of families CE1 (AnAXE1) and CE5 (HjAXE), to reduce acetylation in hybrid aspen. All these transformations improved the saccharification of wood from greenhouse-grown trees. Here, we describe the chemical properties and saccharification potential of the resulting lines grown in a five-year field trial, and one type of them (WP:AnAXE1) in greenhouse conditions. Chemically, the lignocellulose of the field- and greenhouse-field-grown plants slightly differed, but the reductions in acetylation and saccharification improvement of engineered trees were largely maintained in the field. The main novel phenotypic observation in the field was higher lignification in lines with the WP promoter than those with the 35S promoter. Following growth in the field, saccharification glucose yields were higher from most transformed lines than from wild-type (WT) plants with no pretreatment, but there was no improvement in saccharification with acid pretreatment. Thus, acid pretreatment removes most recalcitrance caused by acetylation. We found a complex relationship between acetylation and glucose yields in saccharification without pretreatment, suggesting that other variables, for example, the acetylation pattern, affect recalcitrance. Bigger gains in glucose yields were observed in lines with the 35S promoter than in those with the WP promoter, possibly due to their lower lignin content. However, better lignocellulose saccharification of these lines was offset by a growth penalty and their glucose yield per tree was lower. In a comparison of the best lines with each construct, WP:AnAXE1 provided the highest glucose yield per tree from saccharification, with and without pretreatment, WP:HjAXE yields were similar to those of WT plants, and yields of lines with other constructs were lower. These results show that lignocellulose properties of field-grown trees can be improved by reducing cell wall acetylation using various approaches, but some affect productivity in the field. Thus, better understanding of molecular and physiological consequences of deacetylation is needed to obtain quantitatively better results.

Vessel- and ray-specific monolignol biosynthesis as an approach to engineer fiber-hypolignification and enhanced saccharification in poplar.

Lignin is one of the main factors determining recalcitrance to processing of lignocellulosic biomass towards bio-based materials and fuels. Consequently, wood of plants engineered for low lignin content is typically more amenable to processing. However, lignin-modified plants often exhibit collapsed vessels and associated growth defects. Vessel-specific reintroduction of lignin biosynthesis in dwarfed low-lignin cinnamoyl-CoA reductase1 (ccr1) Arabidopsis mutants using the ProSNBE:AtCCR1 construct overcame the yield penalty while maintaining high saccharification yields, and showed that monolignols can be transported between the different xylem cells acting as 'good neighbors' in Arabidopsis. Here, we translated this research into the bio-energy crop poplar. By expressing ProSNBE:AtCCR1 into CRISPR/Cas9-generated ccr2 poplars, we aimed for vessel-specific lignin biosynthesis to: (i) achieve growth restoration while maintaining high saccharification yields; and (ii) study the existence of 'good neighbors' in poplar wood. Analyzing the resulting ccr2 ProSNBE:AtCCR1 poplars showed that vessels and rays act as good neighbors for lignification in poplar. If sufficient monolignols are produced by these cells, monolignols migrate over multiple cell layers, resulting in a restoration of the lignin amount to wild-type levels. If the supply of monolignols is limited, the monolignols are incorporated into the cell walls of the vessels and rays producing them and their adjoining cells resulting in fiber hypolignification. One such fiber-hypolignified line had 18% less lignin and, despite its small yield penalty, had an increase of up to 71% in sugar release on a plant base upon saccharification.

Acclimation of hydraulic and morphological traits to water deficit delays hydraulic failure during simulated drought in poplar.

The capacity of trees to tolerate and survive increasing drought conditions in situ will depend in part on their ability to acclimate (via phenotypic plasticity) key hydraulic and morphological traits that increase drought tolerance and delay the onset of drought-induced hydraulic failure. However, the effect of water-deficit acclimation in key traits that determine time to hydraulic failure (THF) during extreme drought remains largely untested. We measured key hydraulic and morphological traits in saplings of a hybrid poplar grown under well-watered and water-limited conditions. The time for plants to dry-down to critical levels of water stress (90% loss of stem hydraulic conductance), as well as the relative contribution of drought acclimation in each trait to THF, was simulated using a soil-plant hydraulic model (SurEau). Compared with controls, water-limited plants exhibited significantly lower stem hydraulic vulnerability (P50stem), stomatal conductance and total canopy leaf area (LA). Taken together, adjustments in these and other traits resulted in longer modelled THF in water-limited (~160 h) compared with well-watered plants (~50 h), representing an increase of more than 200%. Sensitivity analysis revealed that adjustment in P50stem and LA contributed the most to longer THF in water-limited plants. We observed a high degree of trait plasticity in poplar saplings in response to water-deficit growth conditions, with decreases in stem hydraulic vulnerability and leaf area playing a key role in delaying the onset of hydraulic failure during a simulated drought event. These findings suggest that understanding the capacity of plants to acclimate to antecedent growth conditions will enable better predictions of plant survivorship during future drought.

Simple, efficient and open-source CRISPR/Cas9 strategy for multi-site genome editing in Populus tremula × alba.

Although the CRISPR/Cas9 system has been successfully used for crop breeding, its application remains limited in forest trees. Here, we describe an efficient gene editing strategy for hybrid poplar, (Populus tremula × alba INRA clone 717-1B4) based on the Golden Gate MoClo cloning. To test the system efficiency for generating single gene mutants, two single guide RNAs (sgRNAs) were designed and incorporated into the MoClo Tool Kit level 2 binary vector with the Cas9 expression cassette to mutate the SHORT ROOT (SHR) gene. Moreover, we also tested its efficiency for introducing mutations in two genes simultaneously by expressing one sgRNA targeting a single site of the YUC4 gene and the other sgRNA targeting the PLT1 gene. For a robust evaluation of the approach, we repeated the strategy to target the LBD12 and LBD4 genes simultaneously, using an independent construct. We generated hairy roots by Agrobacterium rhizogenes-mediated leaf transformation. Sequencing results confirmed the CRISPR/Cas9-mediated mutation in the targeted sites of PtaSHR. Biallelic and homozygous knockout mutations were detected. A deletion spanning both target sites and small insertions/deletions were the most common mutations. Out of the 22 SHR alleles sequenced, 21 were mutated. The phenotype's characterization showed that transgenic roots with biallelic mutations for the SHR gene lacked a defined endodermal single cell layer, suggesting a conserved gene function similar to its homolog in Arabidopsis Arabidopsis thaliana (L.) Heynh. Sequencing results also revealed the high efficiency of the system for generating double mutants. Biallelic mutations for both genes in the yuc4/plt1 and lbd12/lbd4 roots were detected in three (yuc4/plt1) and two (lbd12/lbd4) out of four transgenic roots evaluated. A small deletion or a single nucleotide insertion at the single target site was the most common mutations. This CRISPR/Cas9 strategy arises as a rapid, simple and standardized gene-editing tool to evaluate the gene role in essential developmental programs such as radial cell differentiation of poplar roots.

Leaf and tree responses of young European aspen trees to elevated atmospheric CO2 concentration vary over the season.

Elevated atmospheric CO2 concentration (eCO2) commonly stimulates net leaf assimilation, decreases stomatal conductance and has no clear effect on leaf respiration. However, effects of eCO2 on whole-tree functioning and its seasonal dynamics remain far more uncertain. To evaluate temporal and spatial variability in eCO2 effects, 1-year-old European aspen trees were grown in two treatment chambers under ambient (aCO2, 400 p.p.m.) and elevated (eCO2, 700 p.p.m.) CO2 concentrations during an early (spring 2019) and late (autumn 2018) seasonal experiment. Leaf (net carbon assimilation, stomatal conductance and leaf respiration) and whole-tree (stem growth, sap flow and stem CO2 efflux) responses to eCO2 were measured. Under eCO2, carbon assimilation was stimulated during the early (1.63-fold) and late (1.26-fold) seasonal experiments. Stimulation of carbon assimilation changed over time with largest increases observed in spring when stem volumetric growth was highest, followed by late season down-regulation, when stem volumetric growth ceased. The neutral eCO2 effect on stomatal conductance and leaf respiration measured at leaf level paralleled the unresponsive canopy conductance (derived from sap flow measurements) and stem CO2 efflux measured at tree level. Our results highlight that seasonality in carbon demand for tree growth substantially affects the magnitude of the response to eCO2 at both leaf and whole-tree level.

Early transcriptional response to gravistimulation in poplar without phototropic confounding factors.

In response to gravistimulation under anisotropic light, tree stems showing an active cambium produce reaction wood that redirects the axis of the trees. Several studies have described transcriptomic or proteomic models of reaction wood relative to the opposite wood. However, the mechanisms leading to the formation of reaction wood are difficult to decipher because so many environmental factors can induce various signalling pathways leading to this developmental reprogramming. Using an innovative isotropic device where the phototropic response does not interfere with gravistimulation we characterized the early molecular responses occurring in the stem of poplar after gravistimulation in an isotropic environment, and without deformation of the stem. After 30 min tilting at 35° under anisotropic light, we collected the upper and lower xylems from the inclined stems. Controls were collected from vertical stems. We used a microarray approach to identify differentially expressed transcripts. High-throughput real-time PCR allowed a kinetic experiment at 0, 30, 120 and 180 min after tilting at 35°, with candidate genes. We identified 668 differentially expressed transcripts, from which we selected 153 candidates for additional Fluidigm qPCR assessment. Five candidate co-expression gene clusters have been identified after the kinetic monitoring of the expression of candidate genes. Gene ontology analyses indicate that molecular reprogramming of processes such as 'wood cell expansion', 'cell wall reorganization' and 'programmed cell death' occur as early as 30 min after gravistimulation. Of note is that the change in the expression of different genes involves a fine regulation of gibberellin and brassinosteroid pathways as well as flavonoid and phosphoinositide pathways. Our experimental set-up allowed the identification of genes regulated in early gravitropic response without the bias introduced by phototropic and stem bending responses.

Organic nitrogen nutrition: LHT1.2 protein from hybrid aspen (Populus tremula L. x tremuloides Michx) is a functional amino acid transporter and a homolog of Arabidopsis LHT1.

The contribution of amino acids (AAs) to soil nitrogen (N) fluxes is higher than previously thought. The fact that AA uptake is pivotal for N nutrition in boreal ecosystems highlights plant AA transporters as key components of the N cycle. At the same time, very little is known about AA transport and respective transporters in trees. Tree genomes may contain 13 or more genes encoding the lysine histidine transporter (LHT) family proteins, and this complicates the study of their significance for tree N-use efficiency. With the strategy of obtaining a tool to study N-use efficiency, our aim was to identify and characterize a relevant AA transporter in hybrid aspen (Populus tremula L. x tremuloides Michx.). We identified PtrLHT1.2, the closest homolog of Arabidopsis thaliana (L.) Heynh AtLHT1, which is expressed in leaves, stems and roots. Complementation of a yeast AA uptake mutant verified the function of PtrLHT1.2 as an AA transporter. Furthermore, PtrLHT1.2 was able to fully complement the phenotypes of the Arabidopsis AA uptake mutant lht1 aap5, including early leaf senescence-like phenotype, reduced growth, decreased plant N levels and reduced root AA uptake. Amino acid uptake studies finally showed that PtrLHT1.2 is a high affinity transporter for neutral and acidic AAs. Thus, we identified a functional AtLHT1 homolog in hybrid aspen, which harbors the potential to enhance overall plant N levels and hence increase biomass production. This finding provides a valuable tool for N nutrition studies in trees and opens new avenues to optimizing tree N-use efficiency.

ACAULIS5 Is Required for Cytokinin Accumulation and Function During Secondary Growth of Populus Trees.

In the primary root and young hypocotyl of Arabidopsis, ACAULIS5 promotes translation of SUPPRESSOR OF ACAULIS51 (SAC51) and thereby inhibits cytokinin biosynthesis and vascular cell division. In this study, the relationships between ACAULIS5, SAC51 and cytokinin biosynthesis were investigated during secondary growth of Populus stems. Overexpression of ACAULIS5 from the constitutive 35S promoter in hybrid aspen (Populus tremula × Populus tremuloides) trees suppressed the expression level of ACAULIS5, which resulted in low levels of the physiologically active cytokinin bases as well as their direct riboside precursors in the transgenic lines. Low ACAULIS5 expression and low cytokinin levels of the transgenic trees coincided with low cambial activity of the stem. ACAULIS5 therefore, contrary to its function in young seedlings in Arabidopsis, stimulates cytokinin accumulation and cambial activity during secondary growth of the stem. This function is not derived from maturing secondary xylem tissues as transgenic suppression of ACAULIS5 levels in these tissues did not influence secondary growth. Interestingly, evidence was obtained for increased activity of the anticlinal division of the cambial initials under conditions of low ACAULIS5 expression and low cytokinin accumulation. We propose that ACAULIS5 integrates auxin and cytokinin signaling to promote extensive secondary growth of tree stems.