Integrating multiregulatory analysis reveals the negative regulatory function of miR482a in the response of poplar to canker pathogen infection.

Canker disease caused by the bacterium Lonsdalea populi is one of the most destructive diseases affecting poplar stems. However, the detailed stress response mechanisms of poplar have not been widely characterized. To explore the diverse regulatory RNA landscape and the function of key regulators in poplar subjected to L. populi stress, we integrated time-course experiment with mock-inoculation (CK) and inoculation (IN) with L. populi at the first, third, and sixth day (IN1, IN3, IN6) on Populus × euramericana cv. '74/76' (107), small RNA-seq, whole transcriptome-wide analysis, degradome analysis and transgenic experiments. A total of 98 differentially expressed (DE) miRNA, 17 974 DEmRNA, and 807 DElncRNA were identified in poplar infected by L. populi, presenting dynamic changes over the infection course. Regulatory networks among RNAs were further constructed. Notably, a network centered on ptc-miR482a in CK-vs-IN3 contained most DEGs. We show that miR482a and miR1448 are located in one transcript as a polycistron. Overexpression of pre-miR482a-miR1448 (OX482-1448) and pre-miR482a (OX482) increased poplar susceptibility to canker pathogen with reduced accumulation of reactive oxygen species, while the suppression of miR482a (STTM482) conferred poplar disease resistance. PHA7 was validated as the target of miR482a with degradome sequencing and tobacco transient co-transformation, its expression being downregulated in OX482-1448 and OX482 lines. Additionally, a series of phasiRNAs were triggered by miR482a targeting PHA7, forming regulatory cascades with more RLP, NBS-LRR, and PK genes, further verifying the defense function of miR482a. These findings provide insights for understanding the roles of ncRNAs and regulatory networks involved in poplar immunity.

The ubiquitin E3 ligase RZFP1 affects drought tolerance in poplar by mediating the degradation of the protein phosphatase PP2C-9.

Abscisic acid signaling has been implicated in plant responses to water deficit-induced osmotic stress. However, the underlying molecular mechanism remains unelucidated. This study identified the RING-type E3 ubiquitin ligase RING ZINC FINGER PROTEIN1 (PtrRZFP1) in poplar (Populus trichocarpa), a woody model plant. PtrRZFP1 encodes a ubiquitin E3 ligase that participates in protein ubiquitination. PtrRZFP1 mainly functions in the nucleus and endoplasmic reticulum and is activated by drought and abscisic acid. PtrRZFP1-overexpressing transgenic poplars (35S:PtrRZFP1) showed greater tolerance to drought, whereas PtrRZFP1-knockdown lines (KD-PtrRZFP1) showed greater sensitivity to drought. Under treatment with polyethylene glycol and abscisic acid, PtrRZFP1 promoted the production of NO and H2O2 in stomatal guard cells, ultimately enhancing stomatal closure and improving drought tolerance. Additionally, PtrRZFP1 physically interacted with the clade A Protein Phosphatase 2C protein PtrPP2C-9, a core regulator of abscisic acid signaling, and mediated its ubiquitination and eventual degradation through the ubiquitination-26S proteasome system, indicating that PtrRZFP1 positively regulates the abscisic acid signaling pathway. Furthermore, the PtrPP2C-9-overexpression line was insensitive to abscisic acid and more sensitive to drought than the wild-type plants, whereas the opposite phenotype was observed in 35S:PtrRZFP1 plants. In general, PtrRZFP1 negatively regulates the stability of PtrPP2C-9 to mediate poplar drought tolerance. The results of this study provide a theoretical framework for the targeted breeding of drought-tolerant traits in perennial woody plants.

Genome-wide methylome stability and parental effects in the worldwide distributed Lombardy poplar.

BACKGROUND: Despite the increasing number of epigenomic studies in plants, little is known about the forces that shape the methylome in long-lived woody perennials. The Lombardy poplar offers an ideal opportunity to investigate the impact of the individual environmental history of trees on the methylome. RESULTS: We present the results of three interconnected experiments on Lombardy poplar. In the first experiment, we investigated methylome variability during a growing season and across vegetatively reproduced generations. We found that ramets collected over Europe and raised in common conditions have stable methylomes in symmetrical CG-contexts. In contrast, seasonal dynamics occurred in methylation patterns in CHH context. In the second experiment, we investigated whether methylome patterns of plants grown in a non-parental environment correlate with the parental climate. We did not observe a biological relevant pattern that significantly correlates with the parental climate. Finally, we investigated whether the parental environment has persistent carry-over effects on the vegetative offspring's phenotype. We combined new bud set observations of three consecutive growing seasons with former published bud set data. Using a linear mixed effects analysis, we found a statistically significant but weak short-term, parental carry-over effect on the timing of bud set. However, this effect was negligible compared to the direct effects of the offspring environment. CONCLUSIONS: Genome-wide cytosine methylation patterns in symmetrical CG-context are stable in Lombardy poplar and appear to be mainly the result of random processes. In this widespread poplar clone, methylation patterns in CG-context can be used as biomarkers to infer a common ancestor and thus to investigate the recent environmental history of a specific Lombardy poplar. The Lombardy poplar shows high phenotypic plasticity in a novel environment which enabled this clonal tree to adapt and survive all over the temperate regions of the world.

Ectomycorrhizal symbiosis prepares its host locally and systemically for abiotic cue signaling.

Tree growth and survival are dependent on their ability to perceive signals, integrate them, and trigger timely and fitted molecular and growth responses. While ectomycorrhizal symbiosis is a predominant tree-microbe interaction in forest ecosystems, little is known about how and to what extent it helps trees cope with environmental changes. We hypothesized that the presence of Laccaria bicolor influences abiotic cue perception by Populus trichocarpa and the ensuing signaling cascade. We submitted ectomycorrhizal or non-ectomycorrhizal P. trichocarpa cuttings to short-term cessation of watering or ozone fumigation to focus on signaling networks before the onset of any physiological damage. Poplar gene expression, metabolite levels, and hormone levels were measured in several organs (roots, leaves, mycorrhizas) and integrated into networks. We discriminated the signal responses modified or maintained by ectomycorrhization. Ectomycorrhizas buffered hormonal changes in response to short-term environmental variations systemically prepared the root system for further fungal colonization and alleviated part of the root abscisic acid (ABA) signaling. The presence of ectomycorrhizas in the roots also modified the leaf multi-omics landscape and ozone responses, most likely through rewiring of the molecular drivers of photosynthesis and the calcium signaling pathway. In conclusion, P. trichocarpa-L. bicolor symbiosis results in a systemic remodeling of the host's signaling networks in response to abiotic changes. In addition, ectomycorrhizal, hormonal, metabolic, and transcriptomic blueprints are maintained in response to abiotic cues, suggesting that ectomycorrhizas are less responsive than non-mycorrhizal roots to abiotic challenges.

Comparative transcriptome analysis reveals the importance of phenylpropanoid biosynthesis for the induced resistance of 84K poplar to anthracnose.

BACKGROUND: Poplar anthracnose, which is one of the most important tree diseases, is primarily caused by Colletotrichum gloeosporioides, which has been detected in poplar plantations in China and is responsible for serious economic losses. The characteristics of 84K poplar that have made it one of the typical woody model plants used for investigating stress resistance include its rapid growth, simple reproduction, and adaptability. RESULTS: In this study, we found that the resistance of 84K poplar to anthracnose varied considerably depending on how the samples were inoculated of the two seedlings in each tissue culture bottle, one (84K-Cg) was inoculated for 6 days, whereas the 84K-DCg samples were another seedling inoculated at the 6th day and incubated for another 6 days under the same conditions. It was showed that the average anthracnose spot diameter on 84K-Cg and 84K-DCg leaves was 1.23 ± 0.0577 cm and 0.67 ± 0.1154 cm, respectively. Based on the transcriptome sequencing analysis, it was indicated that the upregulated phenylpropanoid biosynthesis-related genes in 84K poplar infected with C. gloeosporioides, including genes encoding PAL, C4H, 4CL, HCT, CCR, COMT, F5H, and CAD, are also involved in other KEGG pathways (i.e., flavonoid biosynthesis and phenylalanine metabolism). The expression levels of these genes were lowest in 84K-Cg and highest in 84K-DCg. CONCLUSIONS: It was found that PAL-related genes may be crucial for the induced resistance of 84K poplar to anthracnose, which enriched in the phenylpropanoid biosynthesis. These results will provide the basis for future research conducted to verify the contribution of phenylpropanoid biosynthesis to induced resistance and explore plant immune resistance-related signals that may regulate plant defense capabilities, which may provide valuable insights relevant to the development of effective and environmentally friendly methods for controlling poplar anthracnose.

Genome-wide identification and gene expression networks of LBD transcription factors in Populus trichocarpa.

The Lateral Organ Boundaries Domain (LBD) proteins, an exclusive family of transcription factors (TFs) found solely in plants, play pivotal roles in lateral organogenesis, stress adaptation, secondary growth, and hormonal signaling responses. In this study, a total of 55 PtLBD TFs from Populus trichocarpa were identified and systematically classified into two subfamilies, designated as subfamily-I and subfamily-II with seven distinct groups based on phylogenetic analysis. Gene structure detection indicated that the difference of phase numbers linking adjacent exons contribute to the variations in splicing patterns among different PtLBD groups. Numerous transcription factor binding sites and cis-elements pertinent to hormone signaling pathways and stress response mechanisms were identified within the upstream promoter regions of the PtLBD genes. Thirty-five PtLBDs were found to be engaged in either tandem or segmental duplications, and genomic collinearity analysis revealed a stronger alignment between PtLBD genes and eudicots plants compared to their relationship with monocots. GO enrichment and temporal-spatio expression patterns showed that PtLBD7 from subfamily-I and PtLBD20 from subfamily-II, along with other 13 PtLBDs, were involved in plant growth and development biological processes. The multilayered hierarchical gene networks (ML-hGRN) mediated by PtLBD7 and PtLBD20 indicated that PtLBDs were mainly function in poplar growth and stress tolerance through a multifaceted and intricate regulatory machinery. This study lays a solid groundwork for delving deeper into the roles and underlying mechanisms of LBD transcription factors in poplar, specifically those related to plant hormones and stress tolerance.

PagARGOS promotes low-lignin wood formation in poplar.

Wood formation, which occurs mainly through secondary xylem development, is important not only for supplying raw material for the 'ligno-chemical' industry but also for driving the storage of carbon. However, the complex mechanisms underlying the promotion of xylem formation remain to be elucidated. Here, we found that overexpression of Auxin-Regulated Gene involved in Organ Size (ARGOS) in hybrid poplar 84 K (Populus alba × Populus tremula var. glandulosa) enlarged organ size. In particular, PagARGOS promoted secondary growth of stems with increased xylem formation. To gain further insight into how PagARGOS regulates xylem development, we further carried out yeast two-hybrid screening and identified that the auxin transporter WALLS ARE THIN1 (WAT1) interacts with PagARGOS. Overexpression of PagARGOS up-regulated WAT1, activating a downstream auxin response promoting cambial cell division and xylem differentiation for wood formation. Moreover, overexpressing PagARGOS caused not only higher wood yield but also lower lignin content compared with wild-type controls. PagARGOS is therefore a potential candidate gene for engineering fast-growing and low-lignin trees with improved biomass production.

The antagonistic activity of Streptomyces spiroverticillatus (No. HS1) against of poplar canker pathogen Botryosphaeria dothidea.

BACKGROUND: Poplar canker caused by Botryosphaeria dothidea is one of the most severe plant disease of poplars worldwide. In our study, we aimed to investigate the modes of antagonism by fermentation broth supernatant (FBS) of Streptomyces spiroverticillatus HS1 against B. dothidea. RESULTS: In vitro, the strain and FBS of S. spiroverticillatus HS1 significantly inhibited mycelial growth and biomass accumulation, and also disrupted the mycelium morphology of B. dothidea. On the 3rd day after treatment, the inhibition rates of colony growth and dry weight were 80.72% and 52.53%, respectively. In addition, FBS treatment damaged the plasma membrane of B. dothidea based on increased electrical conductivity in the culture medium, and malondialdehyde content of B. dothidea mycelia. Notably, the analysis of key enzymes in glycolysis pathway showed that the activity of hexokinase (HK), phosphofructokinase (PFK), and pyruvate kinase (PK), Ca2+Mg2+-ATPase were significantly increased after FBS treatment. But the glucose contents were significantly reduced, and pyruvate contents were significantly increased in B. dothidea after treatment with FBS. CONCLUSIONS: The inhibitory mechanism of S. spiroverticillatus HS1 against B. dothidea was a complex process, which was associated with multiple levels of mycelial growth, cell membrane structure, material and energy metabolism. The FBS of S. spiroverticillatus HS1 could provide an alternative approach to biological control strategies against B. dothidea.

PdeERF114 recruits PdeWRKY75 to regulate callus formation in poplar by modulating the accumulation of H2 O2 and the relaxation of cell walls.

Callus formation is important for numerous biological processes in plants. Previously, we revealed that the PdeWRKY75-PdeRBOHB module positively regulates hydrogen peroxide (H2 O2 ) accumulation, thereby affecting callus formation in poplar. In this study, we identified and confirmed a transcription factor, PdeERF114, that interacts with PdeWRKY75 both in vitro and in vivo. Gene expression analysis identified both PdeRBOHB and PdeEXPB2 as downstream genes of PdeERF114 and PdeWRKY75. Overexpression (OE) and reduced-expression (RE) transgenic poplar lines for these four genes were generated, and the observation of callus formation was also performed in all plant materials. We demonstrated that PdeERF114 and PdeWRKY75 formed a protein complex and that this complex could bind W-Box motifs in the promoters of PdeRBOHB and PdeEXPB2, thereby positively regulating the expression of PdeRBOHB and PdeEXPB2. The OE/RE transgenic lines for these four genes also showed enhanced/reduced callus formation. Overall, we revealed a novel gene regulatory network for the regulation of callus formation in plants that involves four genes and regulates callus formation through two pathways: the accumulation of H2 O2 in explants and the relaxation of cell walls. In the future, the four genes could be used to enhance transformation effectiveness in genetic engineering.

Unpacking the point of no return under drought in poplar: insight from stem diameter variation.

A specific, robust threshold for drought-induced tree mortality is needed to improve the prediction of forest dieback. Here, we tested the relevance of continuous measurements of stem diameter variations for identifying such a threshold, their relationship with hydraulic and cellular damage mechanisms, and the influence of growth conditions on these relationships. Poplar saplings were grown under well-watered, water-limited, or light-limited conditions and then submitted to a drought followed by rewatering. Stem diameter was continuously measured to investigate two parameters: the percentage loss of diameter (PLD) and the percentage of diameter recovery (DR) following rewatering. Water potentials, stomatal conductance, embolism, and electrolyte leakage were also measured, and light microscopy allowed investigating cell collapse induced by drought. The water release observed through loss of diameter occurred throughout the drought, regardless of growth conditions. Poplars did not recover from drought when PLD reached a threshold and this differed according to growth conditions but remained linked to cell resistance to damage and collapse. Our findings shed new light on the mechanisms of drought-induced tree mortality and indicate that PLD could be a relevant indicator of drought-induced tree mortality, regardless of the growth conditions.

The poplar SWEET1c glucose transporter plays a key role in the ectomycorrhizal symbiosis.

The mutualistic interaction between ectomycorrhizal fungi and trees is characterized by the coordinated exchange of soil nutrients with soluble sugars. Despite the importance of this process, the precise mechanism by which sugars are transported from host roots to colonizing hyphae remains unclear. This study aimed to identify the specific membrane transporters responsible for the unloading of sugars at the symbiotic interface, with a focus on the role of the root Sugars Will Eventually Be Exported Transporter (SWEET) uniporters. Our study used RNA sequencing and quantitative PCR to identify PtaSWEET gene expression in Populus tremula × alba-Laccaria bicolor ectomycorrhizal root tips. Our results suggest that symbiosis-induced PtaSWEET1c is primarily responsible for transporting glucose and sucrose, as demonstrated by the yeast assays. Moreover, we used a promoter-YFP reporter to confirm the localization of the PtaSWEET1c expression in cortical cells of ectomycorrhizal rootlets, supporting its major role in supplying glucose at the symbiotic interface. Furthermore, our observations confirmed the localization of PtaSWEET1c-GFP in the plasma membrane. The inactivation of PtaSWEET1c reduced ectomycorrhizal root formation and 13C translocation to ectomycorrhizal roots. Our findings highlight the crucial role of PtaSWEET1c in facilitating glucose and sucrose transport at the symbiotic interface of Populus-L. bicolor symbiosis.

PagJAZ5 regulates cambium activity through coordinately modulating cytokinin concentration and signaling in poplar.

Wood is resulted from the radial growth paced by the division and differentiation of vascular cambium cells in woody plants, and phytohormones play important roles in cambium activity. Here, we identified that PagJAZ5, a key negative regulator of jasmonate (JA) signaling, plays important roles in enhancing cambium cell division and differentiation by mediating cytokinin signaling in poplar 84K (Populus alba × Populus glandulosa). PagJAZ5 is preferentially expressed in developing phloem and cambium, weakly in developing xylem cells. Overexpression (OE) of PagJAZ5m (insensitive to JA) increased cambium activity and xylem differentiation, while jaz mutants showed opposite results. Transcriptome analyses revealed that cytokinin oxidase/dehydrogenase (CKXs) and type-A response regulators (RRs) were downregulated in PagJAZ5m OE plants. The bioactive cytokinins were significantly increased in PagJAZ5m overexpressing plants and decreased in jaz5 mutants, compared with that in 84K plants. The PagJAZ5 directly interact with PagMYC2a/b and PagWOX4b. Further, we found that the PagRR5 is regulated by PagMYC2a and PagWOX4b and involved in the regulation of xylem development. Our results showed that PagJAZ5 can increase cambium activity and promote xylem differentiation through modulating cytokinin level and type-A RR during wood formation in poplar.

MYC2 regulates stomatal density and water use efficiency via targeting EPF2/EPFL4/EPFL9 in poplar.

Although polyploid plants have lower stomatal density than their diploid counterparts, the molecular mechanisms underlying this difference remain elusive. Here, we constructed a network based on the triploid poplar transcriptome data and triple-gene mutual interaction algorithm and found that PpnMYC2 was related to stomatal development-related genes PpnEPF2, PpnEPFL4, and PpnEPFL9. The interactions between PpnMYC2 and PagJAZs were experimentally validated. PpnMYC2-overexpressing poplar and Arabidopsis thaliana had reduced stomatal density. Poplar overexpressing PpnMYC2 had higher water use efficiency and drought resistance. RNA-sequencing data of poplars overexpressing PpnMYC2 showed that PpnMYC2 promotes the expression of stomatal density inhibitors PagEPF2 and PagEPFL4 and inhibits the expression of the stomatal density-positive regulator PagEPFL9. Yeast one-hybrid system, electrophoretic mobility shift assay, ChIP-qPCR, and dual-luciferase assay were employed to substantiate that PpnMYC2 directly regulated PagEPF2, PagEPFL4, and PagEPFL9. PpnMYC2, PpnEPF2, and PpnEPFL4 were significantly upregulated, whereas PpnEPFL9 was downregulated during stomatal formation in triploid poplar. Our results are of great significance for revealing the regulation mechanism of plant stomatal occurrence and polyploid stomatal density, as well as reducing stomatal density and improving plant water use efficiency by overexpressing MYC2.

LbSakA-mediated phosphorylation of the scaffolding protein LbNoxR in the ectomycorrhizal basidiomycete Laccaria bicolor regulates NADPH oxidase activity, ROS accumulation and symbiosis development.

Ectomycorrhizal symbiosis, which involves mutually beneficial interactions between soil fungi and tree roots, is essential for promoting tree growth. To establish this symbiotic relationship, fungal symbionts must initiate and sustain mutualistic interactions with host plants while avoiding host defense responses. This study investigated the role of reactive oxygen species (ROS) generated by fungal NADPH oxidase (Nox) in the development of Laccaria bicolor/Populus tremula × alba symbiosis. Our findings revealed that L. bicolor LbNox expression was significantly higher in ectomycorrhizal roots than in free-living mycelia. RNAi was used to silence LbNox, which resulted in decreased ROS signaling, limited formation of the Hartig net, and a lower mycorrhizal formation rate. Using Y2H library screening, BiFC and Co-IP, we demonstrated an interaction between the mitogen-activated protein kinase LbSakA and LbNoxR. LbSakA-mediated phosphorylation of LbNoxR at T409, T477 and T480 positively modulates LbNox activity, ROS accumulation and upregulation of symbiosis-related genes involved in dampening host defense reactions. These results demonstrate that regulation of fungal ROS metabolism is critical for maintaining the mutualistic interaction between L. bicolor and P. tremula × alba. Our findings also highlight a novel and complex regulatory mechanism governing the development of symbiosis, involving both transcriptional and posttranslational regulation of gene networks.

Populus MYC2 orchestrates root transcriptional reprogramming of defence pathway to impair Laccaria bicolor ectomycorrhizal development.

The jasmonic acid (JA) signalling pathway plays an important role in the establishment of the ectomycorrhizal symbiosis. The Laccaria bicolor effector MiSSP7 stabilizes JA corepressor JAZ6, thereby inhibiting the activity of Populus MYC2 transcription factors. Although the role of MYC2 in orchestrating plant defences against pathogens is well established, its exact contribution to ECM symbiosis remains unclear. This information is crucial for understanding the balance between plant immunity and symbiotic relationships. Transgenic poplars overexpressing or silencing for the two paralogues of MYC2 transcription factor (MYC2s) were produced, and their ability to establish ectomycorrhiza was assessed. Transcriptomics and DNA affinity purification sequencing were performed. MYC2s overexpression led to a decrease in fungal colonization, whereas its silencing increased it. The enrichment of terpene synthase genes in the MYC2-regulated gene set suggests a complex interplay between the host monoterpenes and fungal growth. Several root monoterpenes have been identified as inhibitors of fungal growth and ECM symbiosis. Our results highlight the significance of poplar MYC2s and terpenes in mutualistic symbiosis by controlling root fungal colonization. We identified poplar genes which direct or indirect control by MYC2 is required for ECM establishment. These findings deepen our understanding of the molecular mechanisms underlying ECM symbiosis.

Suberin deficiency and its effect on the transport physiology of young poplar roots.

The precise functions of suberized apoplastic barriers in root water and nutrient transport physiology have not fully been elucidated. While lots of research has been performed with mutants of Arabidopsis, little to no data are available for mutants of agricultural crop or tree species. By employing a combined set of physiological, histochemical, analytical, and transport physiological methods as well as RNA-sequencing, this study investigated the implications of remarkable CRISPR/Cas9-induced suberization defects in young roots of the economically important gray poplar. While barely affecting overall plant development, contrary to literature-based expectations significant root suberin reductions of up to 80-95% in four independent mutants were shown to not evidently affect the root hydraulic conductivity during non-stress conditions. In addition, subliminal iron deficiency symptoms and increased translocation of a photosynthesis inhibitor as well as NaCl highlight the involvement of suberin in nutrient transport physiology. The multifaceted nature of the root hydraulic conductivity does not allow drawing simplified conclusions such as that the suberin amount must always be correlated with the water transport properties of roots. However, the decreased masking of plasma membrane surface area could facilitate the uptake but also leakage of beneficial and harmful solutes.

Reduced mesophyll conductance under chronic O3 exposure in poplar reflects thicker cell walls and increased subcellular diffusion pathway lengths according to the anatomical model.

Ozone (O3) is one of the most harmful and widespread air pollutants, affecting crop yield and plant health worldwide. There is evidence that O3 reduces the major limiting factor of photosynthesis, namely CO2 mesophyll conductance (gm), but there is little quantitative information of O3-caused changes in key leaf anatomical traits and their impact on gm. We exposed two O3-responsive clones of the economically important tree species Populus × canadensis Moench to 120 ppb O3 for 21 days. An anatomical diffusion model within the leaf was used to analyse the entire CO2 diffusion pathway from substomatal cavities to carboxylation sites and determine the importance of each structural and subcellular component as a limiting factor. gm decreased substantially under O3 and was found to be the most important limitation of photosynthesis. This decrease was mostly driven by an increased cell wall thickness and length of subcellular diffusion pathway caused by altered interchloroplast spacing and chloroplast positioning. By contrast, the prominent leaf integrative trait leaf dry mass per area was neither affected nor related to gm under O3. The observed relationship between gm and anatomy, however, was clone-dependent, suggesting that mechanisms regulating gm may differ considerably between closely related plant lines. Our results confirm the need for further studies on factors constraining gm under stress conditions.

Productive Poplar Genotypes Exhibited Temporally Stable Low Stem Embolism Resistance and Hydraulic Resistance Segmentation at the Stem-Leaf Transition.

Breeding tree genotypes that are both productive and drought-resistant is a primary goal in forestry. However, the relationships between plant hydraulics and yield at the genotype level, and their temporal stabilities, remain unclear. We selected six poplar genotypes from I-101 (Populus alba) × 84 K (P. alba × Popolus tremula var. glandulosa) for experiments in the first and fourth years after planting in a common garden. Measurements included stem embolism resistance, shoot hydraulic resistance and its partitioning between stems and leaves, vessel- and pit-level anatomy, leaf carbon acquisition capacity, carbon allocation to leaves, and aboveground biomass (yield proxy). Significant genetic variations in hydraulic properties and yield were found among genotypes in both years. Productive genotypes had wide vessels, large thin pit membranes, small pit apertures, and shallow pit chambers. Hydraulic resistance was negatively correlated with yield, enabling high stomatal conductance and assimilation rates. Productive genotypes allocated less aboveground carbon and hydraulic resistance to leaves. Temporally stable trade-offs between stem embolism resistance and yield, and between hydraulic segmentation and yield, were identified. These findings highlight the tight link between hydraulic function and yield and suggest that stable trade-offs may challenge breeding poplar genotypes that are both productive and drought-resistant.

PagHAM4a-PagSCL21 and PagHAM4b-PagTCP20 modules positively regulate cambial activity and its differentiation into secondary xylem in poplar.

Stem secondary xylem produced by cambial division and differentiation is the main source of tree biomass. The secondary xylem formation involves a complex transcriptional regulatory network, however, the underlying mechanism is still being explored. Here, we report that PagHAM4a and PagHAM4b are positive regulators of cambial differentiation into secondary xylem in hybrid poplar (Populus alba × Populus glandulosa clone 84K). Overexpression of PagHAM4a and PagHAM4b enhanced cambial activity and increased the number of secondary xylem cells in the stems of poplar. By contrast, single or double mutations of PagHAM4a and PagHAM4b by CRISPR-Cas9 decreased cambial activity, leading to a significant reduction of secondary xylem. Neither overexpression nor mutation of the two genes affected the size of vessels and fibers in xylem. Both PagHAM4a- and PagHAM4b-regulated gene networks were mainly centered in the stage when cambium had just initiated secondary growth, but the molecular networks regulated by the two genes were distinct. Further analysis revealed that PagSCL21 and PagTCP20 are direct targets of PagHAM4a and PagHAM4b, respectively, and their overexpression also promoted cambial differentiation into secondary xylem. Taken together, we identified two novel key regulatory modules in poplar, PagHAM4a-PagSCL21 and PagHAM4b-PagTCP20, which provide new insights into the mechanism of secondary xylem formation in trees.

A strategy for studying epigenetic diversity in natural populations: proof of concept in poplar and oak.

In the last 20 years, several techniques have been developed for quantifying DNA methylation, the most studied epigenetic marks in eukaryotes, including the gold standard method, whole-genome bisulfite sequencing (WGBS). WGBS quantifies genome-wide DNA methylation but has several inconveniences rendering it less suitable for population-scale epigenetic studies. The high cost of deep sequencing and the large amounts of data generated prompted us to seek an alternative approach. Restricting studies to parts of the genome would be a satisfactory alternative had there not been a major limitation: the need to select upstream targets corresponding to differentially methylated regions as targets. Given the need to study large numbers of samples, we propose a strategy for investigating DNA methylation variation in natural populations, taking into account the structural complexity of genomes, their size, and their content in unique coding regions versus repeated regions as transposable elements. We first identified regions of highly variable DNA methylation in a subset of genotypes representative of the biological diversity in the population by WGBS. We then analysed the variations of DNA methylation in these targeted regions at the population level by sequencing capture bisulfite (SeqCapBis). The entire strategy was then validated by applying it to another species. Our strategy was developed as a proof of concept on natural populations of two forest species: Populus nigra and Quercus petraea.