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.

Genotypic differences in systemic root responses to mechanical obstacles.

As roots grow through the soil to forage for water and nutrients, they encounter mechanical obstacles such as patches of dense soil and stones that locally impede root growth. Here, we investigated hitherto poorly understood systemic responses of roots to localised root impedance. Seedlings of two wheat genotypes were grown in hydroponics and exposed to impenetrable obstacles constraining the vertical growth of the primary or a single seminal root. We deployed high-resolution in vivo imaging to quantify temporal dynamics of root elongation rate, helical root movement, and root growth direction. The two genotypes exhibited distinctly different patterns of systemic responses to localised root impedance, suggesting different strategies to cope with obstacles, namely stress avoidance and stress tolerance. Shallower growth of unconstrained seminal roots and more pronounced helical movement of unconstrained primary and seminal roots upon localised root impedance characterised the avoidance strategy shown by one genotype. Stress tolerance to localised root impedance, as exhibited by the other genotype, was indicated by relatively fast elongation of primary roots and steeper seminal root growth. These different strategies highlight that the effects of mechanical obstacles on spatiotemporal root growth patterns can differ within species, which may have major implications for resource acquisition and whole-plant growth.

Quantitative dissection of variations in root growth rate: a matter of cell proliferation or of cell expansion?

Plant organ growth results from cell production and cell expansion. Deciphering the contribution of each of these processes to growth rate is an important issue in developmental biology. Here, we investigated the cellular processes governing root elongation rate, considering two sources of variation: genotype and disturbance by chemicals (NaCl, polyethylene glycol, H2O2, abscisic acid). Exploiting the adventitious rooting capacity of the Populus genus, and using time-lapse imaging under infrared-light, particle image velocimetry, histological analysis, and kinematics, we quantified the cellular processes involved in root growth variation, and analysed the covariation patterns between growth parameters. The rate of cell production by the root apical meristem and the number of dividing cells were estimated in vivo without destructive measurement. We found that the rate of cell division contributed more to the variation in cell production rate than the number of dividing cells. Regardless of the source of variation, the length of the elongation zone was the best proxy for growth rate, summarizing rates of cell production and cell elongation into a single parameter. Our results demonstrate that cell production rate is the main driver of growth rate, whereas elemental elongation rate is a key driver of short-term growth adjustments.

Changes in the epigenome and transcriptome of the poplar shoot apical meristem in response to water availability affect preferentially hormone pathways.

The adaptive capacity of long-lived organisms such as trees to the predicted climate changes, including severe and successive drought episodes, will depend on the presence of genetic diversity and phenotypic plasticity. Here, the involvement of epigenetic mechanisms in phenotypic plasticity toward soil water availability was examined in Populus×euramericana. This work aimed at characterizing (i) the transcriptome plasticity, (ii) the genome-wide plasticity of DNA methylation, and (iii) the function of genes affected by a drought-rewatering cycle in the shoot apical meristem. Using microarray chips, differentially expressed genes (DEGs) and differentially methylated regions (DMRs) were identified for each water regime. The rewatering condition was associated with the highest variations of both gene expression and DNA methylation. Changes in methylation were observed particularly in the body of expressed genes and to a lesser extent in transposable elements. Together, DEGs and DMRs were significantly enriched in genes related to phytohormone metabolism or signaling pathways. Altogether, shoot apical meristem responses to changes in water availability involved coordinated variations in DNA methylation, as well as in gene expression, with a specific targeting of genes involved in hormone pathways, a factor that may enable phenotypic plasticity.

Aboveground overyielding in a mixed temperate forest is not explained by belowground processes.

The relationship between forest productivity and tree species diversity has been described in detail, but the underlying processes have yet to be identified. One important issue is to understand which processes are at the origin of observed aboveground overyielding in some mixed forests. We used a beech-maple plantation exhibiting aboveground overyielding to test whether belowground processes could explain this pattern. Soil cores were collected to determine fine root (FR) biomass and vertical distribution. Correlograms were used to detect spatial arrangement. Near-infrared reflectance spectroscopy was used to identify the tree species proportion in the FR samples and spatial root segregation. An isotopic approach was used to identify water acquisition patterns. The structure and the composition of the ectomycorrhizal fungal community were determined by high-throughput sequencing of DNA in the soil samples. We found no spatial pattern for FR biomass or for its vertical distribution along the gradients. No vertical root segregation was found, as FR density for both species decreased with depth in a similar way. The two species displayed similar vertical water acquisition profiles as well, mainly absorbing water from shallow soil layers; hence, niche differentiation for water acquisition was not highlighted here. Significant alterations in the fungal community compositions were detected in function of the percentage of maple in the vicinity of beech. Our findings do not support the commonly suggested drivers of aboveground overyielding in species-diverse forests and suggest that competition reduction or between-species facilitation of belowground resource acquisition may not explain the observed aboveground overyielding.

Physical root-soil interactions.

Plant root system development is highly modulated by the physical properties of the soil and especially by its mechanical resistance to penetration. The interplay between the mechanical stresses exerted by the soil and root growth is of particular interest for many communities, in agronomy and soil science as well as in biomechanics and plant morphogenesis. In contrast to aerial organs, roots apices must exert a growth pressure to penetrate strong soils and reorient their growth trajectory to cope with obstacles like stones or hardpans or to follow the tortuous paths of the soil porosity. In this review, we present the main macroscopic investigations of soil-root physical interactions in the field and combine them with simple mechanistic modeling derived from model experiments at the scale of the individual root apex.

3D deformation field in growing plant roots reveals both mechanical and biological responses to axial mechanical forces.

Strong regions and physical barriers in soils may slow root elongation, leading to reduced water and nutrient uptake and decreased yield. In this study, the biomechanical responses of roots to axial mechanical forces were assessed by combining 3D live imaging, kinematics and a novel mechanical sensor. This system quantified Young's elastic modulus of intact poplar roots (32MPa), a rapid <0.2 mN touch-elongation sensitivity, and the critical elongation force applied by growing roots that resulted in bending. Kinematic analysis revealed a multiphase bio-mechanical response of elongation rate and curvature in 3D. Measured critical elongation force was accurately predicted from an Euler buckling model, indicating that no biologically mediated accommodation to mechanical forces influenced bending during this short period of time. Force applied by growing roots increased more than 15-fold when buckling was prevented by lateral bracing of the root. The junction between the growing and the mature zones was identified as a zone of mechanical weakness that seemed critical to the bending process. This work identified key limiting factors for root growth and buckling under mechanical constraints. The findings are relevant to crop and soil sciences, and advance our understanding of root growth in heterogeneous structured soils.

The build-up of osmotic stress responses within the growing root apex using kinematics and RNA-sequencing.

Molecular regulation of growth must include spatial and temporal coupling of cell production and cell expansion. The underlying mechanisms, especially under environmental challenge, remain obscure. Spatial patterns of cell processes make the root apex well suited to deciphering stress signaling pathways, and to investigating both processes. Kinematics and RNA-sequencing were used to analyze the immediate growth response of hydroponically grown Populus nigra cuttings submitted to osmotic stress. About 7400 genes and unannotated transcriptionally active regions were differentially expressed between the division and elongation zones. Following the onset of stress, growth decreased sharply, probably due to mechanical effects, before recovering partially. Stress impaired cell expansion over the apex, progressively shortened the elongation zone, and reduced the cell production rate. Changes in gene expression revealed that growth reduction was mediated by a shift in hormone homeostasis. Osmotic stress rapidly elicited auxin, ethylene, and abscisic acid. When growth restabilized, transcriptome remodeling became complex and zone specific, with the deployment of hormone signaling cascades, transcriptional regulators, and stress-responsive genes. Most transcriptional regulations fit growth reduction, but stress also promoted expression of some growth effectors, including aquaporins and expansins Together, osmotic stress interfered with growth by activating regulatory proteins rather than by repressing the machinery of expansive growth.

Length and activity of the root apical meristem revealed in vivo by infrared imaging.

Understanding how cell division and cell elongation influence organ growth and development is a long-standing issue in plant biology. In plant roots, most of the cell divisions occur in a short and specialized region, the root apical meristem (RAM). Although RAM activity has been suggested to be of high importance to understand how roots grow and how the cell cycle is regulated, few experimental and numeric data are currently available. The characterization of the RAM is difficult and essentially based upon cell length measurements through destructive and time-consuming microscopy approaches. Here, a new non-invasive method is described that couples infrared light imaging and kinematic analyses and that allows in vivo measurements of the RAM length. This study provides a detailed description of the RAM activity, especially in terms of cell flux and cell division rate. We focused on roots of hydroponic grown poplars and confirmed our method on maize roots. How the RAM affects root growth rate is studied by taking advantage of the high inter-individual variability of poplar root growth. An osmotic stress was applied and did not significantly affect the RAM length, highlighting its homeostasis in short to middle-term responses. The methodology described here simplifies a lot experimental procedures, allows an increase in the number of individuals that can be taken into account in experiments, and means new experiments can be formulated that allow temporal monitoring of the RAM length.

Phenotypic plasticity toward water regime: response of leaf growth and underlying candidate genes in Populus.

Phenotypic plasticity is considered as an important mechanism for plants to cope with environmental challenges. Leaf growth is one of the first macroscopic processes to be impacted by modification of soil water availability. In this study, we intended to analyze and compare plasticity at different scales. We examined the differential effect of water regime (optimal, moderate water deprivation and recovery) on growth and on the expression of candidate genes in leaves of different growth stages. Candidates were selected to assess components of growth response: abscisic acid signaling, water transport, cell wall modification and stomatal development signaling network. At the tree scale, the four studied poplar hybrids responded similarly to water regime. Meanwhile, leaf growth response was under genotype × environment interaction. Patterns of candidate gene expression enriched our knowledge about their functionality in poplars. For most candidates, transcript levels were strongly structured according to leaf growth performance while response to water regime was clearly dependent on genotype. The use of an index of plasticity revealed that the magnitude of the response was higher for gene expression than for macroscopic traits. In addition, the ranking of poplar genotypes for macroscopic traits well paralleled the one for gene expression.

Plant root growth against a mechanical obstacle: the early growth response of a maize root facing an axial resistance is consistent with the Lockhart model.

Plant root growth is dramatically reduced in compacted soils, affecting the growth of the whole plant. Through a model experiment coupling force and kinematics measurements, we probed the force-growth relationship of a primary root contacting a stiff resisting obstacle, which mimics the strongest soil impedance variation encountered by a growing root. The growth of maize roots just emerging from a corseting agarose gel and contacting a force sensor (acting as an obstacle) was monitored by time-lapse imaging simultaneously to the force. The evolution of the velocity field along the root was obtained from kinematics analysis of the root texture with a particle image velocimetry derived technique. A triangular fit was introduced to retrieve the elemental elongation rate or strain rate. A parameter-free model based on the Lockhart law quantitatively predicts how the force at the obstacle modifies several features of the growth distribution (length of the growth zone, maximal elemental elongation rate and velocity) during the first 10 min. These results suggest a strong similarity of the early growth responses elicited either by a directional stress (contact) or by an isotropic perturbation (hyperosmotic bath).

Genotypic and tissue-specific variation of Populus nigra transcriptome profiles in response to drought.

Climate change is one of the most important challenges for mankind in the far and near future. In this regard, sustainable production of woody crops on marginal land with low water availability is a major challenge to tackle. This dataset is part of an experiment, in which we exposed three genetically differentiated genotypes of Populus nigra originating from contrasting natural habitats to gradually increasing moderate drought. RNA sequencing was performed on fine roots, developing xylem and leaves of those three genotypes under control and moderate drought conditions in order to get a comprehensive dataset on the transcriptional changes at the whole plant level under water limiting conditions. This dataset has already provided insight in the transcriptional control of saccharification potential of the three Populus genotypes under drought conditions and we suggest that our data will be valuable for further in-depth analysis regarding candidate gene identification or, on a bigger scale, for meta-transcriptome analysis.

Monitoring the regulation of gene expression in a growing organ using a fluid mechanics formalism.

BACKGROUND: Technological advances have enabled the accurate quantification of gene expression, even within single cell types. While transcriptome analyses are routinely performed, most experimental designs only provide snapshots of gene expression. Molecular mechanisms underlying cell fate or positional signalling have been revealed through these discontinuous datasets. However, in developing multicellular structures, temporal and spatial cues, known to directly influence transcriptional networks, get entangled as the cells are displaced and expand. Access to an unbiased view of the spatiotemporal regulation of gene expression occurring during development requires a specific framework that properly quantifies the rate of change of a property in a moving and expanding element, such as a cell or an organ segment. RESULTS: We show how the rate of change in gene expression can be quantified by combining kinematics and real-time polymerase chain reaction data in a mechanistic model which considers any organ as a continuum. This framework was applied in order to assess the developmental regulation of the two reference genes Actin11 and Elongation Factor 1-beta in the apex of poplar root. The growth field was determined by time-lapse photography and transcript density was obtained at high spatial resolution. The net accumulation rates of the transcripts of the two genes were found to display highly contrasted developmental profiles. Actin11 showed pulses of up and down regulation in the accelerating and decelerating parts of the growth zone while the dynamic of EF1beta were much slower. This framework provides key information about gene regulation in a developing organ, such as the location, the duration and the intensity of gene induction/repression. CONCLUSIONS: We demonstrated that gene expression patterns can be monitored using the continuity equation without using mutants or reporter constructions. Given the rise of imaging technologies, this framework in our view opens a new way to dissect the molecular basis of growth regulation, even in non-model species or complex structures.

Comparative transcriptomics of drought responses in Populus: a meta-analysis of genome-wide expression profiling in mature leaves and root apices across two genotypes.

BACKGROUND: Comparative genomics has emerged as a promising means of unravelling the molecular networks underlying complex traits such as drought tolerance. Here we assess the genotype-dependent component of the drought-induced transcriptome response in two poplar genotypes differing in drought tolerance. Drought-induced responses were analysed in leaves and root apices and were compared with available transcriptome data from other Populus species. RESULTS: Using a multi-species designed microarray, a genomic DNA-based selection of probesets provided an unambiguous between-genotype comparison. Analyses of functional group enrichment enabled the extraction of processes physiologically relevant to drought response. The drought-driven changes in gene expression occurring in root apices were consistent across treatments and genotypes. For mature leaves, the transcriptome response varied weakly but in accordance with the duration of water deficit. A differential clustering algorithm revealed similar and divergent gene co-expression patterns among the two genotypes. Since moderate stress levels induced similar physiological responses in both genotypes, the genotype-dependent transcriptional responses could be considered as intrinsic divergences in genome functioning. Our meta-analysis detected several candidate genes and processes that are differentially regulated in root and leaf, potentially under developmental control, and preferentially involved in early and long-term responses to drought. CONCLUSIONS: In poplar, the well-known drought-induced activation of sensing and signalling cascades was specific to the early response in leaves but was found to be general in root apices. Comparing our results to what is known in arabidopsis, we found that transcriptional remodelling included signalling and a response to energy deficit in roots in parallel with transcriptional indices of hampered assimilation in leaves, particularly in the drought-sensitive poplar genotype.

Genes and gene clusters related to genotype and drought-induced variation in saccharification potential, lignin content and wood anatomical traits in Populus nigra.

Wood is a renewable resource that can be employed for the production of second generation biofuels by enzymatic saccharification and subsequent fermentation. Knowledge on how the saccharification potential is affected by genotype-related variation of wood traits and drought is scarce. Here, we used three Populus nigra L. genotypes from habitats differing in water availability to (i) investigate the relationships between wood anatomy, lignin content and saccharification and (ii) identify genes and co-expressed gene clusters related to genotype and drought-induced variation in wood traits and saccharification potential. The three poplar genotypes differed in wood anatomy, lignin content and saccharification potential. Drought resulted in reduced cambial activity, decreased vessel and fiber lumina, and increased the saccharification potential. The saccharification potential was unrelated to lignin content as well as to most wood anatomical traits. RNA sequencing of the developing xylem revealed that 1.5% of the analyzed genes were differentially expressed in response to drought, while 67% differed among the genotypes. Weighted gene correlation network analysis identified modules of co-expressed genes correlated with saccharification potential. These modules were enriched in gene ontology terms related to cell wall polysaccharide biosynthesis and modification and vesicle transport, but not to lignin biosynthesis. Among the most strongly saccharification-correlated genes, those with regulatory functions, especially kinases, were prominent. We further identified transcription factors whose transcript abundances differed among genotypes, and which were co-regulated with genes for biosynthesis and modifications of hemicelluloses and pectin. Overall, our study suggests that the regulation of pectin and hemicellulose metabolism is a promising target for improving wood quality of second generation bioenergy crops. The causal relationship of the identified genes and pathways with saccharification potential needs to be validated in further experiments.

Oak genome reveals facets of long lifespan.

Oaks are an important part of our natural and cultural heritage. Not only are they ubiquitous in our most common landscapes1 but they have also supplied human societies with invaluable services, including food and shelter, since prehistoric times2. With 450 species spread throughout Asia, Europe and America3, oaks constitute a critical global renewable resource. The longevity of oaks (several hundred years) probably underlies their emblematic cultural and historical importance. Such long-lived sessile organisms must persist in the face of a wide range of abiotic and biotic threats over their lifespans. We investigated the genomic features associated with such a long lifespan by sequencing, assembling and annotating the oak genome. We then used the growing number of whole-genome sequences for plants (including tree and herbaceous species) to investigate the parallel evolution of genomic characteristics potentially underpinning tree longevity. A further consequence of the long lifespan of trees is their accumulation of somatic mutations during mitotic divisions of stem cells present in the shoot apical meristems. Empirical4 and modelling5 approaches have shown that intra-organismal genetic heterogeneity can be selected for6 and provides direct fitness benefits in the arms race with short-lived pests and pathogens through a patchwork of intra-organismal phenotypes7. However, there is no clear proof that large-statured trees consist of a genetic mosaic of clonally distinct cell lineages within and between branches. Through this case study of oak, we demonstrate the accumulation and transmission of somatic mutations and the expansion of disease-resistance gene families in trees.

Non-invasive Protocol for Kinematic Monitoring of Root Growth under Infrared Light.

Phenotyping the dynamics of root responses to environmental cues is necessary to understand plant acclimation to their environment. Continuous monitoring of root growth is challenging because roots normally grow belowground and are very sensitive to their growth environment. This protocol combines infrared imaging with hydroponic cultivation for kinematic analyses. It allows continuous imaging at fine spatiotemporal resolution and disturbs roots minimally. Examples are provided of how the procedure and materials can be adapted for 3D monitoring and of how environmental stress may be manipulated for experimental purposes.

Analysis of carbohydrates in plants by high-performance anion-exchange chromatography coupled with electrospray mass spectrometry.

A mass spectrometer was coupled to high-performance anion-exchange chromatography (HPAEC) with the help of electrochemical neutralization of the eluent and post-column addition of lithium chloride for carbohydrate analysis. Parallel selective channels (single ion monitoring) were used to decrease the detection limits and separate unresolved peaks. The mass specific detection allowed the simultaneous analysis of a wide range of sugar alcohols, mono-, di- and oligosaccharides. Carbohydrates extracted from leaves of poplar submitted to drought stress were analyzed using pulsed amperometric detection (PAD), then mass spectrometry. It allowed the confirmation of peak attribution and the identification of salicin as a major compound in the extracts. Different responses to water deficit and re-hydration were obtained for several carbohydrates, suggesting different roles in osmoprotection processes.

Developmental and environmental regulation of Aquaporin gene expression across Populus species: divergence or redundancy?

Aquaporins (AQPs) are membrane channels belonging to the major intrinsic proteins family and are known for their ability to facilitate water movement. While in Populus trichocarpa, AQP proteins form a large family encompassing fifty-five genes, most of the experimental work focused on a few genes or subfamilies. The current work was undertaken to develop a comprehensive picture of the whole AQP gene family in Populus species by delineating gene expression domain and distinguishing responsiveness to developmental and environmental cues. Since duplication events amplified the poplar AQP family, we addressed the question of expression redundancy between gene duplicates. On these purposes, we carried a meta-analysis of all publicly available Affymetrix experiments. Our in-silico strategy controlled for previously identified biases in cross-species transcriptomics, a necessary step for any comparative transcriptomics based on multispecies design chips. Three poplar AQPs were not supported by any expression data, even in a large collection of situations (abiotic and biotic constraints, temporal oscillations and mutants). The expression of 11 AQPs was never or poorly regulated whatever the wideness of their expression domain and their expression level. Our work highlighted that PtTIP1;4 was the most responsive gene of the AQP family. A high functional divergence between gene duplicates was detected across species and in response to tested cues, except for the root-expressed PtTIP2;3/PtTIP2;4 pair exhibiting 80% convergent responses. Our meta-analysis assessed key features of aquaporin expression which had remained hidden in single experiments, such as expression wideness, response specificity and genotype and environment interactions. By consolidating expression profiles using independent experimental series, we showed that the large expansion of AQP family in poplar was accompanied with a strong divergence of gene expression, even if some cases of functional redundancy could be suspected.

Gradual soil water depletion results in reversible changes of gene expression, protein profiles, ecophysiology, and growth performance in Populus euphratica, a poplar growing in arid regions.

The responses of Populus euphratica Oliv. plants to soil water deficit were assessed by analyzing gene expression, protein profiles, and several plant performance criteria to understand the acclimation of plants to soil water deficit. Young, vegetatively propagated plants originating from an arid, saline field site were submitted to a gradually increasing water deficit for 4 weeks in a greenhouse and were allowed to recover for 10 d after full reirrigation. Time-dependent changes and intensity of the perturbations induced in shoot and root growth, xylem anatomy, gas exchange, and water status were recorded. The expression profiles of approximately 6,340 genes and of proteins and metabolites (pigments, soluble carbohydrates, and oxidative compounds) were also recorded in mature leaves and in roots (gene expression only) at four stress levels and after recovery. Drought successively induced shoot growth cessation, stomatal closure, moderate increases in oxidative stress-related compounds, loss of CO2 assimilation, and root growth reduction. These effects were almost fully reversible, indicating that acclimation was dominant over injury. The physiological responses were paralleled by fully reversible transcriptional changes, including only 1.5% of the genes on the array. Protein profiles displayed greater changes than transcript levels. Among the identified proteins for which expressed sequence tags were present on the array, no correlation was found between transcript and protein abundance. Acclimation to water deficit involves the regulation of different networks of genes in roots and shoots. Such diverse requirements for protecting and maintaining the function of different plant organs may render plant engineering or breeding toward improved drought tolerance more complex than previously anticipated.