Molecular Advances of Bud Dormancy in Trees.

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. In this review, 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 in the in-depth understanding of bud dormancy and offer insights for future studies.

Core clock genes adjust growth cessation time to day-night switches in poplar.

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.

Spatiotemporal oxygen dynamics in young leaves reveal cyclic hypoxia in plants.

Oxygen is essential for plant growth and development. Hypoxia occurs in plants due to limited oxygen availability following adverse environmental conditions as well in hypoxic niches in otherwise normoxic environments. However, the existence and functional integration of spatiotemporal oxygen dynamics with plant development remains unknown. In animal systems dynamic fluctuations in oxygen availability are known as cyclic hypoxia. In this study, we demonstrate that cyclic fluctuations in internal oxygen levels occur in young emerging leaves of Arabidopsis plants. Cyclic hypoxia in plants is based on a mechanism requiring the ETHYLENE RESPONSE FACTORS type VII (ERFVII) that are central components of the oxygen-sensing machinery in plants. The ERFVII-dependent mechanism allows precise adjustment of leaf growth in response to carbon status and oxygen availability within plant cells. This study thus establishes a functional connection between internal spatiotemporal oxygen dynamics and developmental processes of plants.

The cold-induced factor CBF3 mediates root stem cell activity, regeneration, and developmental responses to cold.

Plant growth and development involve the specification and regeneration of stem cell niches (SCNs). Although plants are exposed to disparate environmental conditions, how environmental cues affect developmental programs and stem cells is not well understood. Root stem cells are accommodated in meristems in SCNs around the quiescent center (QC), which maintains their activity. Using a combination of genetics and confocal microscopy to trace morphological defects and correlate them with changes in gene expression and protein levels, we show that the cold-induced transcription factor (TF) C-REPEAT BINDING FACTOR 3 (CBF3), which has previously been associated with cold acclimation, regulates root development, stem cell activity, and regeneration. CBF3 is integrated into the SHORT-ROOT (SHR) regulatory network, forming a feedback loop that maintains SHR expression. CBF3 is primarily expressed in the root endodermis, whereas the CBF3 protein is localized to other meristematic tissues, including root SCNs. Complementation of cbf3-1 using a wild-type CBF3 gene and a CBF3 fusion with reduced mobility show that CBF3 movement capacity is required for SCN patterning and regulates root growth. Notably, cold induces CBF3, affecting QC activity. Furthermore, exposure to moderate cold around 10°C-12°C promotes root regeneration and QC respecification in a CBF3-dependent manner during the recuperation period. By contrast, CBF3 does not appear to regulate stem cell survival, which has been associated with recuperation from more acute cold (∼4°C). We propose a role for CBF3 in mediating the molecular interrelationships among the cold response, stem cell activity, and development.

JGI Plant Gene Atlas: an updateable transcriptome resource to improve functional gene descriptions across the plant kingdom.

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 updateable data resource consisting of transcript abundance assays spanning 18 diverse species. To integrate across these diverse genotypes, we analyzed expression profiles, built gene clusters that exhibited tissue/condition specific expression, and tested for transcriptional response to environmental queues. We discovered extensive phylogenetically constrained and condition-specific expression profiles for genes without any 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 differentially expressed genes, track orthologs across the Gene Atlas plants, graphically represent co-expressed genes, and visualize gene ontology and pathway enrichments.

DNA hypomethylation of the host tree impairs interaction with mutualistic ectomycorrhizal fungus.

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.

Concentration-dependent transcriptional switching through a collective action of cis-elements.

Gene expression specificity of homeobox transcription factors has remained paradoxical. WUSCHEL activates and represses CLAVATA3 transcription at lower and higher concentrations, respectively. We use computational modeling and experimental analysis to investigate the properties of the cis-regulatory module. We find that intrinsically each cis-element can only activate CLAVATA3 at a higher WUSCHEL concentration. However, together, they repress CLAVATA3 at higher WUSCHEL and activate only at lower WUSCHEL, showing that the concentration-dependent interactions among cis-elements regulate both activation and repression. Biochemical experiments show that two adjacent functional cis-elements bind WUSCHEL with higher affinity and dimerize at relatively lower levels. Moreover, increasing the distance between cis-elements prolongs WUSCHEL monomer binding window, resulting in higher CLAVATA3 activation. Our work showing a constellation of optimally spaced cis-elements of defined affinities determining activation and repression thresholds in regulating CLAVATA3 transcription provides a previously unknown mechanism of cofactor-independent regulation of transcription factor binding in mediating gene expression specificity.

Overexpression of a SOC1-Related Gene Promotes Bud Break in Ecodormant Poplars.

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.

FLOWERING LOCUS T2 Promotes Shoot Apex Development and Restricts Internode Elongation via the 13-Hydroxylation Gibberellin Biosynthesis Pathway in Poplar.

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.

LHY2 Integrates Night-Length Information to Determine Timing of Poplar Photoperiodic Growth.

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.

Engineering Tree Seasonal Cycles of Growth Through Chromatin Modification.

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.

Photoperiodic Regulation of Shoot Apical Growth in Poplar.

Woody perennials adapt their genetic traits to local climate conditions. Day length plays an essential role in the seasonal growth of poplar trees. When photoperiod falls below a given critical day length, poplars undergo growth cessation and bud set. A leaf-localized mechanism of photoperiod measurement triggers the transcriptional modulation of a long distance signaling molecule, FLOWERING LOCUS T (FT). This molecule targets meristem function giving rise to these seasonal responses. Studies over the past decade have identified conserved orthologous genes involved in photoperiodic flowering in Arabidopsis that regulate poplar vegetative growth. However, phenological and molecular examination of key photoperiod signaling molecules reveals functional differences between these two plant model systems suggesting alternative components and/or regulatory mechanisms operating during poplar vegetative growth. Here, we review current knowledge and provide new data regarding the molecular components of the photoperiod measuring mechanism that regulates annual growth in poplar focusing on main achievements and new perspectives.

Cytokinin stabilizes WUSCHEL by acting on the protein domains required for nuclear enrichment and transcription.

Concentration-dependent transcriptional regulation and the spatial regulation of transcription factor levels are poorly studied in plant development. WUSCHEL, a stem cell-promoting homeodomain transcription factor, accumulates at a higher level in the rib meristem than in the overlying central zone, which harbors stem cells in the shoot apical meristems of Arabidopsis thaliana. The differential accumulation of WUSCHEL in adjacent cells is critical for the spatial regulation and levels of CLAVATA3, a negative regulator of WUSCHEL transcription. Earlier studies have revealed that DNA-dependent dimerization, subcellular partitioning and protein destabilization control WUSCHEL protein levels and spatial accumulation. Moreover, the destabilization of WUSCHEL may also depend on the protein concentration. However, the roles of extrinsic spatial cues in maintaining differential accumulation of WUS are not understood. Through transient manipulation of hormone levels, hormone response patterns and analysis of the receptor mutants, we show that cytokinin signaling in the rib meristem acts through the transcriptional regulatory domains, the acidic domain and the WUSCHEL-box, to stabilize the WUS protein. Furthermore, we show that the same WUSCHEL-box functions as a degron sequence in cytokinin deficient regions in the central zone, leading to the destabilization of WUSCHEL. The coupled functions of the WUSCHEL-box in nuclear retention as described earlier, together with cytokinin sensing, reinforce higher nuclear accumulation of WUSCHEL in the rib meristem. In contrast a sub-threshold level may expose the WUSCHEL-box to destabilizing signals in the central zone. Thus, the cytokinin signaling acts as an asymmetric spatial cue in stabilizing the WUSCHEL protein to lead to its differential accumulation in neighboring cells, which is critical for concentration-dependent spatial regulation of CLAVATA3 transcription and meristem maintenance. Furthermore, our work shows that cytokinin response is regulated independently of the WUSCHEL function which may provide robustness to the regulation of WUSCHEL concentration.

Environmentally Sensitive Molecular Switches Drive Poplar Phenology.

Boreal and temperate woody perennials are highly adapted to their local climate, which delimits the length of the growing period. Moreover, seasonal control of growth-dormancy cycles impacts tree productivity and geographical distribution. Therefore, traits related to phenology are of great interest to tree breeders and particularly relevant in the context of global warming. The recent application of transcriptional profiling and genetic association studies to poplar species has provided a robust molecular framework for investigating molecules with potential links to phenology. The environment dictates phenology by modulating the expression of endogenous molecular switches, the identities of which are currently under investigation. This review outlines the current knowledge of these molecular switches in poplar and covers several perspectives concerning the environmental control of growth-dormancy cycles. In the process, we highlight certain genetic pathways which are affected by short days, low temperatures and cold-induced signaling.

Overexpression of DEMETER, a DNA demethylase, promotes early apical bud maturation in poplar.

The transition from active growth to dormancy is critical for the survival of perennial plants. We identified a DEMETER-like (CsDML) cDNA from a winter-enriched cDNA subtractive library in chestnut (Castanea sativa Mill.), an economically and ecologically important species. Next, we characterized this DNA demethylase and its putative ortholog in the more experimentally tractable hybrid poplar (Populus tremula × alba), under the signals that trigger bud dormancy in trees. We performed phylogenetic and protein sequence analysis, gene expression profiling, and 5-methyl-cytosine methylation immunodetection studies to evaluate the role of CsDML and its homolog in poplar, PtaDML6. Transgenic hybrid poplars overexpressing CsDML were produced and analysed. Short days and cold temperatures induced CsDML and PtaDML6. Overexpression of CsDML accelerated short-day-induced bud formation, specifically from Stages 1 to 0. Buds acquired a red-brown coloration earlier than wild-type plants, alongside with the up-regulation of flavonoid biosynthesis enzymes and accumulation of flavonoids in the shoot apical meristem and bud scales. Our data show that the CsDML gene induces bud formation needed for the survival of the apical meristem under the harsh conditions of winter.

Chilling-responsive DEMETER-LIKE DNA demethylase mediates in poplar bud break.

Annual dormancy-growth cycle is a developmental and physiological process essential for the survival of deciduous trees in temperate and boreal forests. Seasonal control of shoot growth in woody perennials requires specific genetic programmes responding to environmental signals. The environmental-controlled mechanisms that regulate the shift between winter dormancy and the growth-promoting genetic programmes are still unknown. Here, we show that dynamics in genomic DNA methylation levels are involved in the regulation of dormancy-growth cycle in poplar. The reactivation of growth in the apical shoot during bud break process in spring is preceded by a progressive reduction of genomic DNA methylation in apex tissue. The induction in apex tissue of a chilling-dependent poplar DEMETER-LIKE 10 (PtaDML10) DNA demethylase precedes shoot growth reactivation. Transgenic poplars showing downregulation of PtaDML8/10 caused delayed bud break. Genome-wide transcriptome and methylome analysis and data mining revealed that the gene targets of DEMETER-LIKE-dependent DNA demethylation are genetically associated with bud break. These data point to a chilling-dependent DEMETER-like DNA demethylase mechanisms being involved in the shift from winter dormancy to a condition that precedes shoot apical vegetative growth in poplar.

Real-time monitoring of PtaHMGB activity in poplar transactivation assays.

BACKGROUND: Precise control of gene expression is essential to synchronize plant development with the environment. In perennial plants, transcriptional regulation remains poorly understood, mainly due to the long time required to perform functional studies. Transcriptional reporters based on luciferase have been useful to study circadian and diurnal regulation of gene expression, both by transcription factors and chromatin remodelers. The high mobility group proteins are considered transcriptional chaperones that also modify the chromatin architecture. They have been found in several species, presenting in some cases a circadian expression of their mRNA or protein. RESULTS: Transactivation experiments have been shown as a powerful and fast method to obtain information about the potential role of transcription factors upon a certain reporter. We designed and validated a luciferase transcriptional reporter using the 5' sequence upstream ATG of Populus tremula × alba LHY2 gene. We showed the robustness of this reporter line under long day and continuous light conditions. Moreover, we confirmed that pPtaLHY2::LUC activity reproduces the accumulation of PtaLHY2 mRNA. We performed transactivation studies by transient expression, using the reporter line as a genetic background, unraveling a new function of a high mobility group protein in poplar, which can activate the PtaLHY2 promoter in a gate-dependent manner. We also showed PtaHMGB2/3 needs darkness to produce that activation and exhibits an active degradation after dawn, mediated by the 26S proteasome. CONCLUSIONS: We generated a stable luciferase reporter poplar line based on the circadian clock gene PtaLHY2, which can be used to investigate transcriptional regulation and signal transduction pathway. Using this reporter line as a genetic background, we established a methodology to rapidly assess potential regulators of diurnal and circadian rhythms. This tool allowed us to demonstrate that PtaHMGB2/3 promotes the transcriptional activation of our reporter in a gate-dependent manner. Moreover, we added new information about the PtaHMGB2/3 protein regulation along the day. This methodology can be easily adapted to other transcription factors and reporters.

DNA-dependent homodimerization, sub-cellular partitioning, and protein destabilization control WUSCHEL levels and spatial patterning.

The homeodomain transcription factor WUSCHEL (WUS) promotes stem cell maintenance in inflorescence meristems of Arabidopsis thaliana WUS, which is synthesized in the rib meristem, migrates and accumulates at lower levels in adjacent cells. Maintenance of WUS protein levels and spatial patterning distribution is not well-understood. Here, we show that the last 63-aa stretch of WUS is necessary for maintaining different levels of WUS protein in the rib meristem and adjacent cells. The 63-aa region contains the following transcriptional regulatory domains: the acidic region, the WUS-box, which is conserved in WUS-related HOMEOBOX family members, and the ethylene-responsive element binding factor-associated amphiphilic repression (EAR-like) domain. Our analysis reveals that the opposing functions of WUS-box, which is required for nuclear retention, and EAR-like domain, which participates in nuclear export, are necessary to maintain higher nuclear levels of WUS in cells of the rib meristem and lower nuclear levels in adjacent cells. We also show that the N-terminal DNA binding domain, which is required for both DNA binding and homodimerization, along with the homodimerization sequence located in the central part of the protein, restricts WUS from spreading excessively and show that the homodimerization is critical for WUS function. Our analysis also reveals that a higher level of WUS outside the rib meristem leads to protein destabilization, suggesting a new tier of regulation in WUS protein regulation. Taken together our data show that processes that influence WUS protein levels and spatial distribution are highly coupled to its transcriptional activity.

Threshold-dependent transcriptional discrimination underlies stem cell homeostasis.

Transcriptional mechanisms that underlie the dose-dependent regulation of gene expression in animal development have been studied extensively. However, the mechanisms of dose-dependent transcriptional regulation in plant development have not been understood. In Arabidopsis shoot apical meristems, WUSCHEL (WUS), a stem cell-promoting transcription factor, accumulates at a higher level in the rib meristem and at a lower level in the central zone where it activates its own negative regulator, CLAVATA3 (CLV3). How WUS regulates CLV3 levels has not been understood. Here we show that WUS binds a group of cis-elements, cis- regulatory module, in the CLV3-regulatory region, with different affinities and conformations, consisting of monomers at lower concentration and as dimers at a higher level. By deleting cis elements, manipulating the WUS-binding affinity and the homodimerization threshold of cis elements, and manipulating WUS levels, we show that the same cis elements mediate both the activation and repression of CLV3 at lower and higher WUS levels, respectively. The concentration-dependent transcriptional discrimination provides a mechanistic framework to explain the regulation of CLV3 levels that is critical for stem cell homeostasis.

Adaptive geometric tessellation for 3D reconstruction of anisotropically developing cells in multilayer tissues from sparse volumetric microscopy images.

The need for quantification of cell growth patterns in a multilayer, multi-cellular tissue necessitates the development of a 3D reconstruction technique that can estimate 3D shapes and sizes of individual cells from Confocal Microscopy (CLSM) image slices. However, the current methods of 3D reconstruction using CLSM imaging require large number of image slices per cell. But, in case of Live Cell Imaging of an actively developing tissue, large depth resolution is not feasible in order to avoid damage to cells from prolonged exposure to laser radiation. In the present work, we have proposed an anisotropic Voronoi tessellation based 3D reconstruction framework for a tightly packed multilayer tissue with extreme z-sparsity (2-4 slices/cell) and wide range of cell shapes and sizes. The proposed method, named as the 'Adaptive Quadratic Voronoi Tessellation' (AQVT), is capable of handling both the sparsity problem and the non-uniformity in cell shapes by estimating the tessellation parameters for each cell from the sparse data-points on its boundaries. We have tested the proposed 3D reconstruction method on time-lapse CLSM image stacks of the Arabidopsis Shoot Apical Meristem (SAM) and have shown that the AQVT based reconstruction method can correctly estimate the 3D shapes of a large number of SAM cells.