Effective phytoremediation of trace element-contaminated soils requires crops capable of simultaneously accumulating high metal loads while maintaining cellular stress tolerance. In this study, the capacity of the overexpression of the transcription factor TEMPRANILLO-LIKE 2 (TEML2) in conferring tolerance to Zinc (Zn) and Cadmium (Cd) in Populus was investigated. Using long-term Zn and Cd stress, TEML2 overexpressing (TEML2 OX) plants showed improved tolerance and greater metal accumulation than wild-type plants. Transcriptomic analysis revealed that TEML2 OX plants did not activate a generic metal stress response. Instead, TEML2 appears to regulate distinct stress-adaptive metabolic programs. Under Zn excess, it prioritized homeostatic restoration, ROS scavenging, and targeted lipidome restructuring to prevent lipid peroxidation. Conversely, under Cd toxicity, TEML2 shifted towards photosynthetic maintenance, hydraulic integrity, and energy conservation. Despite these divergent trajectories, the downregulation of Polyamines Oxidases (PAO), pointing to a reduced polyamine catabolism, was identified as the convergent metabolic linchpin of the TEML2 OX in response to Zn and Cd excess. Exogenous polyamine application unequivocally resulted in tolerant metal-excluder phenotypes, indicating that the interaction between TEML2 OX and PAO downregulation triggers alternative pathways. Ultimately, TEML2 was identified as a master regulatory hub, providing a comprehensive genetic blueprint for engineering high-biomass crops to remediate complex, multi-metal contaminated environments.

Wood formation and tree growth depend on the precise spatiotemporal distribution of auxin, yet how woody perennials dynamically regulate local auxin pools remains poorly understood. Here, we demonstrate that the tonoplast-localized transporters WAT1 and WAT2 drive cambial activity, secondary growth, and wood patterning in poplar by controlling stem free auxin availability. Unlike annual models such as Arabidopsis, simultaneous disruption of WAT1 and WAT2 in poplar induces severe dwarfism and marked wood defects, yielding smaller, denser vessel elements, all of which were rescued by genetic complementation. Loss of WAT function depleted stem indole-3-acetic acid (IAA) by up to 96%, without affecting the shoot-resident IAA levels. WAT proteins accumulate in cambial vacuoles in an auxin-dependent manner, peaking during night cycles aligned with maximal auxin responses. Depleting active auxin in wat1 wat2 double mutants triggers broad transcriptomic reprogramming that suppresses cambial proliferation and impairs cell wall remodeling via altered pectin methylesterification. Quantitative data-driven computer model further reveals that WAT proteins function as an intracellular rheostat regulating auxin dynamics across light-dark cycles. Our findings establish that dynamic intracellular compartmentalization fine-tunes local auxin availability in the cambium to ensure robust tree patterning and growth.

BACKGROUND:Drought significantly affects forests and woody crops by limiting their growth, increasing their susceptibility to diseases, and reducing productivity. Wood anatomical plasticity is a crucial adaptive mechanism that enables trees to cope with fluctuations in water availability. During severe drought, trees develop more and narrower vessels, enhancing hydraulic safety and reducing the risk of embolism. However, the molecular regulation of vessel formation is still not well understood. RESULTS:Using single-nucleus transcriptomics, we have generated a cell type-specific gene expression map of the mature poplar stem under well-watered and drought conditions. Our findings reveal extensive gene expression reprogramming in xylem-forming cells, with changes in auxin homeostasis identified as a key mechanism for anatomical adaptation. Specifically, we show that poplar WAT1-like genes control vessel spatial patterning. Additionally, the downregulation of WAT1-like gene expression in the dividing cells of the vascular cambium and the upregulation of MP-like gene expression in cells undergoing early vessel differentiation facilitate the formation of secondary xylem with narrower and more numerous vessels under drought. Furthermore, the wat2 mutant exhibits greater drought tolerance than wild-type trees, underscoring its potential for developing drought-resilient tree varieties. CONCLUSIONS:This study provides the first single-nucleus transcriptomic map of hybrid poplar stems under severe drought, uncovering auxin-driven hormonal networks that regulate xylem plasticity and enhance drought tolerance. These insights provide valuable targets for improving resilience in poplar and other woody species.

Understanding how perennial plants such as trees perceive, integrate and memorize environmental stresses like water deficit is crucial in the context of climate change. We investigated short-term and transannual memory of water deficit in cambium derived tissues of poplars ( Populus spp .) using two contrasting genotypes and four genetically modified epitypes with altered DNA methylation machinery. We found persistent changes in hormone profiles, gene expression and DNA methylation one week after stress relief, consistent with a molecular short-term stress memory. These signatures revealed distinct adaptive strategies between genotypes and marked variability between epitypes, demonstrating that both genetic and epigenetic backgrounds drive stress memory. Trees already exposed to water deficit in Year 1 showed distinct physiological and molecular responses when subjected to a second water deficit in Year 2, supporting the persistence of a long-term trans-annual memory possibly involved in tree priming. The higher molecular plasticity observed in the intrinsically more sensitive genotype contrasted with the higher stability observed in the intrinsically more tolerant genotype. The number of memory genes was limited, but the results suggested that stable drought-induced DNA methylation changes, particularly in the CG context, contribute to long-term stress imprinting. Our findings highlight the cambium as a reservoir for stress memory in trees and suggest that DNA methylation dynamics shaped by genetic predisposition, acting through cis- and trans-regulatory routes, may help fine-tuning growth vs. survival strategies across longer time frames. These insights open perspectives for harnessing epigenetic variation in tree breeding and management under increasing drought frequency. ### Competing Interest Statement The authors have declared no competing interest. Agence Nationale de la Recherche, ANR-17-CE32-0009

Trees in temperate and boreal latitudes synchronize their growth-dormancy cycles with seasonal environmental variations to ensure their survival over the years. Dormancy control is crucial during winter when plants cease growth and establish buds to protect their apical meristems from cold temperatures. To overcome endormancy, initiate bud break, and restore growth, plants must be exposed to a specific duration of chilling, referred to as the chilling requirement, which is species- and ecotype-dependent. In this study, we study the novel roles of two TEMPRANILLO-like genes (TEML1 and TEML2) in the annual cycle of poplar. We demonstrated that Populus TEML genes are regulated by photoperiod, cold temperatures and the circadian clock, and they are induced in buds by short days and chilling treatment. Notably, their function diverges from the role of its Arabidopsis ortholog AtTEM, which regulates FLOWERING LOCUS T (FT) transcription and the photoperiodic flowering transcription. Transcriptomic analysis of apical buds during short days and chilling treatment revealed that the overexpression of TEML1 and TEML2 accelerate dormancy release by modulating the expression of dormancy regulators and growth-promoting genes.

Poplar trees use photoperiod as a precise seasonal indicator, synchronizing plant phenology with the environment. Daylength cue determines FLOWERING LOCUS T 2 ( FT2 ) daily expression, crucial for shoot apex development and establishment of the annual growing period. However, limited evidence exists for the molecular factors controlling FT2 transcription and the conservation with the photoperiodic control of Arabidopsis flowering. We demonstrate that FT2 expression mediates growth cessation response quantitatively, and we provide a minimal data-driven model linking core clock genes to FT2 daily levels. GIGANTEA ( GI ) emerges as a critical inducer of the FT2 activation window, time-bound by TIMING OF CAB EXPRESSION ( TOC1 ) and LATE ELONGATED HYPOCOTYL ( LHY2 ) repressions. CRISPR/Cas9 loss-of-function lines validate these roles, identifying TOC1 as a long-sought FT2 repressor. Additionally, model simulations predict that FT2 downregulation upon daylength shortening results from a progressive narrowing of this activation window, driven by the phase shift observed in the preceding clock genes. This circadian-mediated mechanism enables poplar to exploit FT2 levels as an accurate daylength-meter.

Seasonal bud dormancy in perennial woody plants is a crucial and intricate process that is vital for the survival and development of plants. Over the past few decades, significant advancements have been made in understanding many features of bud dormancy, particularly in model species, where certain molecular mechanisms underlying this process have been elucidated. We provide an overview of recent molecular progress in understanding bud dormancy in trees, with a specific emphasis on the integration of common signaling and molecular mechanisms identified across different tree species. Additionally, we address some challenges that have emerged from our current understanding of bud dormancy and offer insights for future studies. We present an overview of recent molecular advances in bud dormancy in trees, with a particular focus on the integration of common molecular mechanisms identified among different tree species.

Gene functional descriptions offer a crucial line of evidence for candidate genes underlying trait variation. Conversely, plant responses to environmental cues represent important resources to decipher gene function and subsequently provide molecular targets for plant improvement through gene editing. However, biological roles of large proportions of genes across the plant phylogeny are poorly annotated. Here we describe the Joint Genome Institute (JGI) Plant Gene Atlas, an update able data resource consisting of transcript abundance assays spanning 18 diverse species. To integrate across these diverse genotypes, we anal yzed e xpression pr ofiles, b uilt gene c lusters that exhibited tissue / condition specific expression, and tested for transcriptional response to environmental queues. We discovered extensive phylogenetically constrained and condition-specific expression profiles f or genes without an y previously documented functional annotation. Such conserved expression patterns and tightly co-expressed gene clusters let us assign expression derived additional biological information to 64 495 genes with otherwise unknown functions. The ever-expanding Gene Atlas resource is available at JGI Plant Gene Atlas ( https://plantgeneatlas.jgi.doe.gov ) and Phytozome ( https://phytozome .jgi.doe .gov/), providing bulk access to data and user-specified queries of gene sets. Combined, these web interfaces let users access differentiall y e xpressed genes, track orthologs across the Gene Atlas plants, graphically represent co-expressed genes, and visualize gene ontology and pathway enrichments.

Ectomycorrhizas are an intrinsic component of tree nutrition and responses to environmental variations. How epigenetic mechanisms might regulate these mutualistic interactions is unknown. By manipulating the level of expression of the chromatin remodeler DECREASE IN DNA METHYLATION 1 (DDM1) and two demethylases DEMETER-LIKE (DML) in Populus tremula × Populus alba lines, we examined how host DNA methylation modulates multiple parameters of the responses to root colonization with the mutualistic fungus Laccaria bicolor. We compared the ectomycorrhizas formed between transgenic and wild-type (WT) trees and analyzed their methylomes and transcriptomes. The poplar lines displaying lower mycorrhiza formation rate corresponded to hypomethylated overexpressing DML or RNAi-ddm1 lines. We found 86 genes and 288 transposable elements (TEs) differentially methylated between WT and hypomethylated lines (common to both OX-dml and RNAi-ddm1) and 120 genes/1441 TEs in the fungal genome suggesting a host-induced remodeling of the fungal methylome. Hypomethylated poplar lines displayed 205 differentially expressed genes (cis and trans effects) in common with 17 being differentially methylated (cis). Our findings suggest a central role of host and fungal DNA methylation in the ability to form ectomycorrhizas including not only poplar genes involved in root initiation, ethylene and jasmonate-mediated pathways, and immune response but also terpenoid metabolism.

Lignin biosynthesis in the sclerenchyma cells is strictly controlled by a complex network of genetic and environmental signals. In the last decades, the transcriptional regulation of lignin synthesis in woody species has been established. However, the role of microRNA-mediated post-transcriptional modulation in secondary cell wall biosynthesis remains poorly understood. Here, we identified a microRNA, miR828, involved in the regulation specific to lignin biosynthesis during stem development in Populus tomentosa Carr. miR828 is preferentially expressed in the secondary vascular tissues during stem development. Two MYB genes (MYB171 and MYB011) were validated as direct targets of miR828 by degradome analysis and green fluorescent protein signal detection. Overexpression of miR828 in poplar downregulated genes for lignin biosynthesis, resulting in reduced lignin content in cell walls. Conversely, suppression of miR828 in plants by the short tandem target mimics elevated the expression of lignin biosynthetic genes and increased lignin deposition. We further revealed that poplar MYB171, as the most abundant miR828 target in the stem, is a positive regulator for lignin biosynthesis. Transient expression assays showed that both MYB171 and MYB011 activated PAL1 and CCR2 transcription, whereas the introduction of miR828 significantly suppressed their expression that was induced by MYB171 or MYB011. Collectively, our results demonstrate that the miR828-MYBs module precisely regulates lignin biosynthesis during the stem development in P. tomentosa through transcriptional and post-transcriptional manners.

The adaptation and survival of boreal and temperate perennials relies on the precise demarcation of the growing season. Seasonal growth and development are defined by day length and temperature signals. Under long-day conditions in spring, poplar FLOWERING LOCUS T2 (FT2) systemically induces shoot growth. In contrast, FT2 downregulation induced by autumnal short days triggers growth cessation and bud set. However, the molecular role of FT2 in local and long-range signaling is not entirely understood. In this study, the CRISPR/Cas9 editing tool was used to generate FT2 loss of function lines of hybrid poplar. Results indicate that FT2 is essential to promote shoot apex development and restrict internode elongation under conditions of long days. The application of bioactive gibberellins (GAs) to apical buds in FT2 loss of function lines was able to rescue bud set. Expression analysis of GA sensing and metabolic genes and hormone quantification revealed that FT2 boosts the 13-hydroxylation branch of the GA biosynthesis pathway in the shoot apex. Paclobutrazol treatment of WT leaves led to limited internode growth in the stem elongation zone. In mature leaves, FT2 was found to control the GA 13-hydroxylation pathway by increasing GA2ox1 and reducing GA3ox2 expression, causing reduced GA1 levels. We here show that in poplar, the FT2 signal promotes shoot apex development and restricts internode elongation through the GA 13-hydroxylation pathway.

ABSTRACTGene functional descriptions, which are typically derived from sequence similarity to experimentally validated genes in a handful of model species, offer a crucial line of evidence when searching for candidate genes that underlie trait variation. Plant responses to environmental cues, including gene expression regulatory variation, represent important resources for understanding gene function and crucial targets for plant improvement through gene editing and other biotechnologies. However, even after years of effort and numerous large-scale functional characterization studies, biological roles of large proportions of protein coding genes across the plant phylogeny are poorly annotated. Here we describe the Joint Genome Institute (JGI) Plant Gene Atlas, a public and updateable data resource consisting of transcript abundance assays from 2,090 samples derived from 604 tissues or conditions across 18 diverse species. We integrated across these diverse conditions and genotypes by analyzing expression profiles, building gene clusters that exhibited tissue/condition specific expression, and testing for transcriptional modulation in response to environmental queues. For example, we discovered extensive phylogenetically constrained and condition-specific expression profiles across many gene families and genes without any functional annotation. Such conserved expression patterns and other tightly co-expressed gene clusters let us assign expression derived functional descriptions to 64,620 genes with otherwise unknown functions. The ever-expanding Gene Atlas resource is available at JGI Plant Gene Atlas (https://plantgeneatlas.jgi.doe.gov) and Phytozome (https://phytozome-next.jgi.doe.gov), providing bulk access to data and user-specified queries of gene sets. Combined, these web interfaces let users access differentially expressed genes, track orthologs across the Gene Atlas plants, graphically represent co-expressed genes, and visualize gene ontology and pathway enrichments.

Perennial species in the boreal and temperate regions are subject to extreme annual variations in light and temperature. They precisely adapt to seasonal changes by synchronizing cycles of growth and dormancy with external cues. Annual dormancy–growth transitions and flowering involve factors that integrate environmental and endogenous signals. MADS-box transcription factors have been extensively described in the regulation of Arabidopsis flowering. However, their participation in annual dormancy–growth transitions in trees is minimal. In this study, we investigate the function of MADS12, a Populus tremula × alba SUPPRESSOR OF CONSTANS OVEREXPRESSION 1 (SOC1)-related gene. Our gene expression analysis reveals that MADS12 displays lower mRNA levels during the winter than during early spring and mid-spring. Moreover, MADS12 activation depends on the fulfillment of the chilling requirement. Hybrid poplars overexpressing MADS12 show no differences in growth cessation and bud set, while ecodormant plants display an early bud break, indicating that MADS12 overexpression promotes bud growth reactivation. Comparative expression analysis of available bud break-promoting genes reveals that MADS12 overexpression downregulates the GIBBERELLINS 2 OXIDASE 4 (GA2ox4), a gene involved in gibberellin catabolism. Moreover, the mid-winter to mid-spring RNAseq profiling indicates that MADS12 and GA2ox4 show antagonistic expression during bud dormancy release. Our results support MADS12 participation in the reactivation of shoot meristem growth during ecodormancy and link MADS12 activation and GA2ox4 downregulation within the temporal events that lead to poplar bud break.

Day length is a key indicator of seasonal information that determines major patterns of behavior in plants and animals. Photoperiodism has been described in plants for about 100 years, but the underlying molecular mechanisms of day length perception and signal transduction in many systems are not well understood. In trees, photoperiod perception plays a major role in growth cessation during the autumn as well as activating the resumption of shoot growth in the spring, both processes controlled by FLOWERING LOCUS T2 (FT2) expression levels and critical for the survival of perennial plants over winter [1-4]. It has been shown that the conserved role of poplar orthologs to Arabidopsis CONSTANS (CO) directly activates FT2 expression [1, 5]. Overexpression of poplar CO is, however, not sufficient to sustain FT2 expression under short days [5], pointing to the presence of an additional short-day-dependent FT2 repression pathway in poplar. We find that night length information is transmitted via the expression level of a poplar clock gene, LATE ELONGATED HYPOCOTYL 2 (LHY2), which controls FT2 expression. Repression of FT2 is a function of the night extension and LHY2 expression level. We show that LHY2 is necessary and sufficient to activate night length repressive signaling. We propose that the photoperiodic control of shoot growth in poplar involves a balance between FT2 activating and repressing pathways. Our results show that poplar relies on night length measurement to determine photoperiodism through interaction between light signaling pathways and the circadian clock.

In temperate and boreal regions, perennial trees arrest cell division in their meristematic tissues during winter dormancy until environmental conditions become appropriate for their renewed growth. Release from the dormant state requires exposure to a period of chilling temperatures similar to the vernalization required for flowering in Arabidopsis. Over the past decade, genomic DNA (gDNA) methylation and transcriptome studies have revealed signatures of chromatin regulation during active growth and winter dormancy. To date, only a few chromatin modification genes, as candidate regulators of these developmental stages, have been functionally characterized in trees. In this work, we summarize the major findings of the chromatin-remodeling role during growth-dormancy cycles and we explore the transcriptional profiling of vegetative apical bud and stem tissues during dormancy. Finally, we discuss genetic strategies designed to improve the growth and quality of forest trees.

EDITORIAL article Front. Plant Sci., 11 October 2019Sec. Plant Biotechnology Volume 10 - 2019 | https://doi.org/10.3389/fpls.2019.01187

Forest trees are long-lived organisms subject to repeated environmental constraints throughout their long lifetimes. They have developed various mechanisms enabling them to cope with fluctuating environmental conditions during their life span, and to survive to current climate change. Epigenetics has recently emerged as a powerful set of mechanisms regulating various developmental processes, plant growth and responses to environmental variations. Such epigenetic mechanisms, which may remain stable along tree life or across generations, constitute a source of rapid phenotypic variations potentially improving adaptation of the plants in situations in which naturally occurring mutations are very rare. In this review, we summarize recent advances in forest tree epigenomics. We first draw the particularities of trees and the available (epi) genomics resources and strategies. Then, we discuss the potential contributions of epigenetics to cope with global climate change and regulate various developmental processes, such as developmental transitions during the annual cycle, phenotypic plasticity in response to environmental variations and stress memory, as well as local adaptation. Finally, we propose some challenges for forest management and highlighted the need to take epigenetics into account in forest tree breeding strategies.