Osmotic adjustments support growth of poplar cultured cells under high concentrations of carbohydrates.

KEY MESSAGE: Poplar callus maintained a specific difference in osmotic potential with respect to media when supplemented with different carbohydrate concentrations. This balance in osmotic potential guaranteed the growth capacity. Osmotic stress is caused by several abiotic factors such as drought, salinity, or freezing. However, the threshold of osmotic potential that allows the growth under stress conditions has not been thoroughly studied. In this study, different levels of osmotic stress in Populus alba (L.) callus have been induced with the addition of mannitol or sorbitol in the medium (from 0 to 500 mM). The key factor for preserving the growth was observed to be the restoration of a constant difference in osmotic potential between callus and medium for all the tested conditions. The osmotic adjustments were primarily achieved with the uptake of mannitol or sorbitol from the media considering their chemical properties instead of their biological functions. The decrease in water content (from - 1 to - 10% after 21 days) and mineral elements, such as potassium, calcium, and magnesium, together with the alterations in cell morphology, did not show negative effects on growth. The activity of sorbitol dehydrogenase was detected for the first time in poplar (+ 4.7 U l-1 in callus treated with sorbitol compared to control callus). This finding suggested the importance of choosing carefully the molecules used to exert osmotic stress for separating the dual function of carbohydrates in osmotic adjustments and cell metabolism.

Molecular Mechanism of Vegetative Growth Advantage in Allotriploid Populus.

Allotriploid poplar has a prominent vegetative growth advantage that impacts dramatically on lumber yield. The growth regulation is complex which involves abundant genes, metabolic and signaling pathways, while the information about the functional control process is very little. We used high-throughput sequencing and physiological index measurement to obtain a global overview of differences between allotriploid and diploid Populus. The genes related to plant growth advantage show a higher expression compared to diploid, and most of them are revolved around hormones, photosynthesis and product accumulation. Thus, allotriploid Populus showed more efficient photosynthesis, carbon fixation, sucrose and starch synthesis, and metabolism as well as augmented biosynthesis of auxin, cytokinin, and gibberellin. These data enable the connection of metabolic processes, signaling pathways, and specific gene activity, which will underpin the development of network models to elucidate the process of triploid Populus advantage growth.

Structural features in tension wood and distribution of wall polymers in the G-layer of in vitro grown poplars.

Under the effect of disturbances, like unbalanced stem, but also during normal development, poplar trees can develop a specific secondary xylem, called "tension wood" (TW), which is easily identifiable by the presence of a gelatinous layer in the secondary cell walls (SCW) of the xylem fibers. Since TW formation was mainly performed on 2-year-old poplar models, an in vitro poplar that produces gelatinous fibers (G-fibers) while offering the same experimental advantages as herbaceous plants has been developed. Using specific cell wall staining techniques, wood structural features and lignin/cellulose distribution were both detailed in cross-sections obtained from the curved stem part of in vitro poplars. A supposed delay in the SCW lignification process in the G-fibers, along with the presence of a G-layer, could be observed in the juvenile plants. Moreover, in this G-layer, the immunolabeling of various polymers carried out in the SCW of TW has allowed detecting crystalline cellulose, arabinogalactans proteins, and rhamnogalacturonans I; however, homogalacturonans, xylans, and xyloglucans could not be found. Interestingly, extensins were detected in this typical adaptative or stress-induced structure. These observations were corroborated by a quantitation of the immunorecognized polymer distribution using gold particle labeling. In conclusion, the in vitro poplar model seems highly convenient for TW studies focusing on the implementation of wall polymers that provide the cell wall with greater plasticity in adapting to the environment.

Analysis of Orthologous SECONDARY WALL-ASSOCIATED NAC DOMAIN1 (SND1) Promotor Activity in Herbaceous and Woody Angiosperms.

SECONDARY WALL-ASSOCIATED NAC DOMAIN1 (SND1) is a master regulator of fibre secondary wall deposition in Arabidopsis thaliana (Arabidopsis), with homologs in other angiosperms and gymnosperms. However, it is poorly understood to what extent the fibre-specific regulation of the SND1 promoter, and that of its orthologs, is conserved between diverged herbaceous and woody lineages. We performed a reciprocal reporter gene analysis of orthologous SND1 promoters from Arabidopsis (AthSND1), Eucalyptus grandis (EgrNAC61) and Populus alba × P. grandidentata (PagWND1A) relative to secondary cell wall-specific Cellulose Synthase4 (CesA4) and CesA7 promoters, in both a non-woody (Arabidopsis) and a woody (poplar) system. ß-glucuronidase (GUS) reporter analysis in Arabidopsis showed that the SND1 promoter was active in vascular tissues as previously reported and showed interfascicular and xylary fibre-specific expression in inflorescence stems, while reporter constructs of the woody plant-derived promoters were partial to the (pro)cambium-phloem and protoxylem. In transgenic P. tremula × P. alba plants, all three orthologous SND1 promoters expressed the GUS reporter similarly and preferentially in developing secondary xylem, ray parenchyma and cork cambium. Ours is the first study to reciprocally test orthologous SND1 promoter specificity in herbaceous and woody species, revealing diverged regulatory functions in the herbaceous system.

Populus NST/SND orthologs are key regulators of secondary cell wall formation in wood fibers, phloem fibers and xylem ray parenchyma cells.

Wood fibers form thick secondary cell wall (SCW) in xylem tissues to give mechanical support to trees. NAC SECONDARY WALL THICKENING PROMOTING FACTOR3/SECONDARY WALL-ASSOCIATED NAC DOMAIN PROTEIN 1 (NST3/SND1) and NST1 were identified as master regulators of SCW formation in xylem fiber cells in the model plant Arabidopsis thaliana. In Populus species, four NST/SND orthologs have been conserved and coordinately control SCW formation in wood fibers and phloem fibers. However, it remains to be elucidated whether SCW formation in other xylem cells, such as ray parenchyma cells and vessel elements, is regulated by NST/SND orthologs in poplar. We knocked out all NST/SND genes in hybrid aspen using the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 nuclease (Cas9) system and investigated the detailed histological appearance of stem tissues in the knockout mutants. Observation by light microscopy and transmission electron microscopy showed that SCW was severely suppressed in wood fibers, phloem fibers and xylem ray parenchyma cells in the knockout mutants. Although almost all wood fibers lacked SCW, some fiber cells formed thick cell walls. The irregularly cell wall-forming fibers retained primary wall and SCW, and were mainly located in the vicinity of vessel elements. Field emission-scanning electron microscope observation showed that there were no apparent differences in the structural features of pits such as the shape and size between irregularly SCW-forming wood fibers in the knockout mutants and normal wood fibers in wild-type. Cell wall components such as cellulose, hemicellulose and lignin were deposited in the cell wall of irregularly SCW-forming wood fibers in quadruple mutants. Our results indicate that four NST/SND orthologs are master switches for SCW formation in wood fibers, xylem ray parenchyma cells and phloem fibers in poplar, while SCW is still formed in limited wood fibers, which are located at the region adjacent to vessel elements in the knockout mutants.

Computational models evaluating the impact of sieve plates and radial water exchange on phloem pressure gradients.

The sugar conducting phloem in angiosperms is a high resistance pathway made up of sieve elements bounded by sieve plates. The high resistance generated by sieve plates may be a trade-off for promoting quick sealing in the event of injury. However, previous modeling efforts have demonstrated a wide variation in the contribution of sieve plates towards total sieve tube resistance. In the current study, we generated high resolution scanning electron microscope images of sieve plates from balsam poplar and integrated them into a mathematical model using Comsol Multiphysics software. We found that sieve plates contribute upwards of 85% towards total sieve tube resistance. Utilizing the Navier-Stokes equations, we found that oblong pores may create over 50% more resistance in comparison with round pores of the same area. Although radial water flows in phloem sieve tubes have been previously considered, their impact on alleviating pressure gradients has not been fully studied. Our novel simulations find that radial water flow can reduce pressure requirements by half in comparison with modeled sieve tubes with no radial permeability. We discuss the implication that sieve tubes may alleviate pressure requirements to overcome high resistances by regulating their membrane permeability along the entire transport pathway.

Ethylene signaling induces gelatinous layers with typical features of tension wood in hybrid aspen.

The phytohormone ethylene impacts secondary stem growth in plants by stimulating cambial activity, xylem development and fiber over vessel formation. We report the effect of ethylene on secondary cell wall formation and the molecular connection between ethylene signaling and wood formation. We applied exogenous ethylene or its precursor 1-aminocyclopropane-1-carboxylic acid (ACC) to wild-type and ethylene-insensitive hybrid aspen trees (Populus tremula × tremuloides) and studied secondary cell wall anatomy, chemistry and ultrastructure. We furthermore analyzed the transcriptome (RNA Seq) after ACC application to wild-type and ethylene-insensitive trees. We demonstrate that ACC and ethylene induce gelatinous layers (G-layers) and alter the fiber cell wall cellulose microfibril angle. G-layers are tertiary wall layers rich in cellulose, typically found in tension wood of aspen trees. A vast majority of transcripts affected by ACC are downstream of ethylene perception and include a large number of transcription factors (TFs). Motif-analyses reveal potential connections between ethylene TFs (Ethylene Response Factors (ERFs), ETHYLENE INSENSITIVE 3/ETHYLENE INSENSITIVE3-LIKE1 (EIN3/EIL1)) and wood formation. G-layer formation upon ethylene application suggests that the increase in ethylene biosynthesis observed during tension wood formation is important for its formation. Ethylene-regulated TFs of the ERF and EIN3/EIL1 type could transmit the ethylene signal.

High temperature exposure did not affect induced 2n pollen viability in Populus.

High temperature exposure is widely used as a physical mutagenic agent to induce 2n gametes in Populus. However, whether high temperature exposure affects induced 2n pollen viability remains unknown. To clarify whether high temperature exposure affected the induced 2n pollen viability, 2n pollen induced by 38 and 41 °C temperatures, pollen morphology, 2n pollen germination in vitro, and crossing induced 2n pollen with normal gametes to produce a triploid was, based on observations of meiosis, conducted in Populus canescens. We found that the dominant meiotic stages (F = 56.6, p < .001) and the treatment duration (F = 21.4, p < .001) significantly affected the occurrence rate of induced 2n pollen. A significant decrease in pollen production and an increase in aborted pollen were observed (p < .001). High temperature sometimes affected in ectexine deposition and some narrow furrows were also analysed via details of ectexine structure. However, no significant difference in 2n pollen germination rate was observed between natural 2n pollen (26.7%) and high-temperature-induced 2n pollen (26.2%), and 42 triploids were created by crossing high-temperature-induced 2n pollen, suggesting that 38 and 41 °C temperatures exposure will not result in dysfunctional induced 2n pollen.

Inositol Hexakis Phosphate is the Seasonal Phosphorus Reservoir in the Deciduous Woody Plant Populus alba L.

Seasonal recycling of nutrients is an important strategy for deciduous perennials. Deciduous perennials maintain and expand their nutrient pools by the autumn nutrient remobilization and the subsequent winter storage throughout their long life. Phosphorus (P), one of the most important elements in living organisms, is remobilized from senescing leaves during autumn in deciduous trees. However, it remains unknown how phosphate is stored over winter. Here we show that in poplar trees (Populus alba L.), organic phosphates are accumulated in twigs from late summer to winter, and that IP6 (myo-inositol-1,2,3,4,5,6-hexakis phosphate: phytic acid) is the primary storage form. IP6 was found in high concentrations in twigs during winter and quickly decreased in early spring. In parenchyma cells of winter twigs, P was associated with electron-dense structures, similar to globoids found in seeds of higher plants. Various other deciduous trees were also found to accumulate IP6 in twigs during winter. We conclude that IP6 is the primary storage form of P in poplar trees during winter, and that it may be a common strategy for seasonal P storage in deciduous woody plants.

Plasticity, elasticity, and adhesion energy of plant cell walls: nanometrology of lignin loss using atomic force microscopy.

The complex organic polymer, lignin, abundant in plants, prevents the efficient extraction of sugars from the cell walls that is required for large scale biofuel production. Because lignin removal is crucial in overcoming this challenge, the question of how the nanoscale properties of the plant cell ultrastructure correlate with delignification processes is important. Here, we report how distinct molecular domains can be identified and how physical quantities of adhesion energy, elasticity, and plasticity undergo changes, and whether such quantitative observations can be used to characterize delignification. By chemically processing biomass, and employing nanometrology, the various stages of lignin removal are shown to be distinguished through the observed morphochemical and nanomechanical variations. Such spatially resolved correlations between chemistry and nanomechanics during deconstruction not only provide a better understanding of the cell wall architecture but also is vital for devising optimum chemical treatments.

Immunoelectron Microscopy of Cryofixed and Freeze-Substituted Plant Tissues.

Cryofixation and freeze-substitution techniques provide excellent preservation of plant ultrastructure. The advantage of cryofixation is not only in structural preservation, as seen in the smooth plasma membrane, but also in the speed in arresting cell activity. Immunoelectron microscopy reveals the subcellular localization of molecules within cells. Immunolabeling in combination with cryofixation and freeze-substitution techniques provides more detailed information on the immunoelectron-microscopic localization of molecules in the plant cell than can be obtained from chemically fixed tissues. Here, we introduce methods for immunoelectron microscopy of cryofixed and freeze-substituted plant tissues.

Drought-induced xylem pit membrane damage in aspen and balsam poplar.

Drought induces an increase in a tree's vulnerability to a loss of its hydraulic conductivity in many tree species, including two common in western Canada, trembling aspen (Populus tremuloides) and balsam poplar (Populus balsamifera). Termed 'cavitation fatigue' or 'air-seeding fatigue', the mechanism of this phenomenon is not well understood, but hypothesized to be a result of damage to xylem pit membranes. To examine the validity of this hypothesis, the effect of drought on the porosity of pit membranes in aspen and balsam poplar was investigated. Controlled drought and bench dehydration treatments were used to induce fatigue and scanning electron microscopy (SEM) was used to image pit membranes for relative porosity evaluations from air-dried samples after ethanol dehydration. A significant increase in the diameter of the largest pore was found in the drought and dehydration treatments of aspen, while an increase in the percentage of porous pit membranes was found in the dehydration treatments of both species. Additionally, the location of the largest pore per pit membrane was observed to tend toward the periphery of the membrane.

Sexual competition affects biomass partitioning, carbon-nutrient balance, Cd allocation and ultrastructure of Populus cathayana females and males exposed to Cd stress.

Although increasing attention has been paid to plant adaptation to soil heavy metal contamination, competition and neighbor effects have been largely overlooked, especially in dioecious plants. In this study, we investigated growth as well as biochemical and ultrastructural responses of Populus cathayana Rehder females and males to cadmium (Cd) stress under different sexual competition patterns. The results showed that competition significantly affects biomass partitioning, photosynthetic capacity, leaf and root ultrastructure, Cd accumulation, the contents of polyphenols, and structural and nonstructural carbohydrates. Compared with single-sex cultivation, plants of opposite sexes exposed to sexual competition accumulated more Cd in tissues and their growth was more strongly inhibited, indicating enhanced Cd toxicity under sexual competition. Under intrasexual competition, females showed greater Cd accumulation, more serious damage at the ultrastructural level and greater reduction in physiological activity than under intersexual competition, while males performed better under intrasexual competition than under intersexual competition. Males improved the female microenvironment by greater Cd uptake and lower resource consumption under intersexual competition. These results demonstrate that the sex of neighbor plants and competition affect sexual differences in growth and in key physiological processes under Cd stress. The asymmetry of sexual competition highlighted here might regulate population structure, and spatial segregation and phytoremediation potential of both sexes in P. cathayana growing in heavy metal-contaminated soils.

Opto-nanomechanical spectroscopic material characterization.

The non-destructive, simultaneous chemical and physical characterization of materials at the nanoscale is an essential and highly sought-after capability. However, a combination of limitations imposed by Abbe diffraction, diffuse scattering, unknown subsurface, electromagnetic fluctuations and Brownian noise, for example, have made achieving this goal challenging. Here, we report a hybrid approach for nanoscale material characterization based on generalized nanomechanical force microscopy in conjunction with infrared photoacoustic spectroscopy. As an application, we tackle the outstanding problem of spatially and spectrally resolving plant cell walls. Nanoscale characterization of plant cell walls and the effect of complex phenotype treatments on biomass are challenging but necessary in the search for sustainable and renewable bioenergy. We present results that reveal both the morphological and compositional substructures of the cell walls. The measured biomolecular traits are in agreement with the lower-resolution chemical maps obtained with infrared and confocal Raman micro-spectroscopies of the same samples. These results should prove relevant in other fields such as cancer research, nanotoxicity, and energy storage and production, where morphological, chemical and subsurface studies of nanocomposites, nanoparticle uptake by cells and nanoscale quality control are in demand.

Knocking Down of Isoprene Emission Modifies the Lipid Matrix of Thylakoid Membranes and Influences the Chloroplast Ultrastructure in Poplar.

Isoprene is a small lipophilic molecule with important functions in plant protection against abiotic stresses. Here, we studied the lipid composition of thylakoid membranes and chloroplast ultrastructure in isoprene-emitting (IE) and nonisoprene-emitting (NE) poplar (Populus × canescens). We demonstrated that the total amount of monogalactosyldiacylglycerols, digalactosyldiacylglycerols, phospholipids, and fatty acids is reduced in chloroplasts when isoprene biosynthesis is blocked. A significantly lower amount of unsaturated fatty acids, particularly linolenic acid in NE chloroplasts, was associated with the reduced fluidity of thylakoid membranes, which in turn negatively affects photosystem II photochemical efficiency. The low photosystem II photochemical efficiency in NE plants was negatively correlated with nonphotochemical quenching and the energy-dependent component of nonphotochemical quenching. Transmission electron microscopy revealed alterations in the chloroplast ultrastructure in NE compared with IE plants. NE chloroplasts were more rounded and contained fewer grana stacks and longer stroma thylakoids, more plastoglobules, and larger associative zones between chloroplasts and mitochondria. These results strongly support the idea that in IE species, the function of this molecule is closely associated with the structural organization and functioning of plastidic membranes.

Mapping symplasmic fields at the shoot apical meristem using iontophoresis and membrane potential measurements.

Microinjections of fluorescent dyes have revealed that the shoot apical meristem (SAM) is dynamically partitioned into symplasmic fields (SFs), implying that plasmodesmata (Pd) are held shut at specific locations in the proliferating cellular matrix. The SFs are integrated into a coherent morphogenetic unit by exchange of morphogens and transcription factors via gating Pd between adjacent SFs, and by ligand-receptor interactions that operate across the extracellular space. We describe a method for the real-time mapping of SF in the SAM by iontophoresis and membrane potential measurements.

Immunolabelling of intervessel pits for polysaccharides and lignin helps in understanding their hydraulic properties in Populus tremula × alba.

BACKGROUND AND AIMS: The efficiency and safety functions of xylem hydraulics are strongly dependent on the pits that connect the xylem vessels. However, little is known about their biochemical composition and thus about their hydraulic properties. In this study, the distribution of the epitopes of different wall components (cellulose, hemicelluloses, pectins and lignins) was analysed in intervessel pits of hybrid poplar (Populus tremula × alba). METHODS: Immunogold labelling with transmission electron microscopy was carried out with a set of antibodies raised against different epitopes for each wall polysaccharide type and for lignins. Analyses were performed on both immature and mature vessels. The effect of sap ionic strength on xylem conductance was also tested. KEY RESULTS: In mature vessels, the pit membrane (PM) was composed of crystalline cellulose and lignins. None of the hemicellulose epitopes were found in the PM. Pectin epitopes in mature vessels were highly concentrated in the annulus, a restricted area of the PM, whereas they were initially found in the whole PM in immature vessels. The pit border also showed a specific labelling pattern, with higher cellulose labelling compared with the secondary wall of the vessel. Ion-mediated variation of 24 % was found for hydraulic conductance. CONCLUSIONS: Cellulose microfibrils, lignins and annulus-restricted pectins have different physicochemical properties (rigidity, hydrophobicity, porosity) that have different effects on the hydraulic functions of the PM, and these influence both the hydraulic efficiency and vulnerability to cavitation of the pits, including ion-mediated control of hydraulic conductance. Impregnation of the cellulose microfibrils of the PM with lignins, which have low wettability, may result in lower cavitation pressure for a given pore size and thus help to explain the vulnerability of this species to cavitation.

Method for automatically identifying spectra of different wood cell wall layers in Raman imaging data set.

The technique of Raman spectroscopic imaging is finding ever-increasing applications in the field of wood science for its ability to provide spatial and spectral information about the sample. On the basis of the acquired Raman imaging data set, it is possible to determine the distribution of chemical components in various wood cell wall layers. However, the Raman imaging data set often contains thousands of spectra measured at hundreds or even thousands of individual frequencies, which results in difficulties accurately and quickly extracting all of the spectra within a specific morphological region of wood cell walls. To address this issue, the authors propose a new method to automatically identify Raman spectra of different cell wall layers on the basis of principal component analysis (PCA) and cluster analysis. A Raman imaging data set collected from a 55.5 µm × 47.5 µm cross-section of poplar tension wood was analyzed. Several thousand spectra were successfully classified into five groups in accordance with different morphological regions, namely, cell corner (CC), compound middle lamella (CML), secondary wall (SW), gelatinous layer (G-layer), and cell lumen. Their corresponding average spectra were also calculated. In addition, the relationship between different characteristic peaks in the obtained Raman spectra was estimated and it was found that the peak at 1331 cm(-1) is more related to lignin rather than cellulose. Not only can this novel method provide a convenient and accurate procedure for identifying the spectra of different cell wall layers in a Raman imaging data set, but it also can bring new insights into studying the morphology and topochemistry in wood cell walls.

Ultrastructure of plant leaf cuticles in relation to sample preparation as observed by transmission electron microscopy.

The leaf cuticular ultrastructure of some plant species has been examined by transmission electron microscopy (TEM) in only few studies. Attending to the different cuticle layers and inner structure, plant cuticles have been grouped into six general morphological types. With the aim of critically examining the effect of cuticle isolation and preparation for TEM analysis on cuticular ultrastructure, adaxial leaf cuticles of blue-gum eucalypt, grey poplar, and European pear were assessed, following a membrane science approach. The embedding and staining protocols affected the ultrastructure of the cuticles analysed. The solubility parameter, surface tension, and contact angles with water of pure Spurr's and LR-White resins were within a similar range. Differences were however estimated for resin : solvent mixtures, since Spurr's resin is combined with acetone and LR-White resin is mixed with ethanol. Given the composite hydrophilic and lipophilic nature of plant cuticles, the particular TEM tissue embedding and staining procedures employed may affect sample ultrastructure and the interpretation of the results in physicochemical and biological terms. It is concluded that tissue preparation procedures may be optimised to facilitate the observation of the micro- and nanostructure of cuticular layers and components with different degrees of polarity and hydrophobicity.

Using confocal Raman microscopy to real-time monitor poplar cell wall swelling and dissolution during ionic liquid pretreatment.

The ionic liquids (ILs) are recognized as the potential solvents for the pretreatment of lignocellulosic materials before biomass conversion. However, little knowledge of how the cell wall of biomass responds to the IL locally and dynamically during the pretreatment is available. In the current work, the process of IL pretreatment of poplar using 1-ethyl-3-methylimidazolium acetate ([C2 mim][OAc]) was real-time monitored on a cellular level by employing confocal Raman microscopy. The results showed that the biomass dissolution during the IL pretreatment can be clearly divided into two stages: (1) slow penetration of IL, and (2) rapid dissolution of lignin and carbohydrates. In this case, the onset of the dissolution of these compositions occurred only after the cell wall of biomass swelled to a certain extent. Because the first stage was a slow process which determined the process reaction rate, it can be deduced that enhancing the penetration capacity of IL was crucial for improving the pretreatment efficiency. Based on the obtained results, a model was proposed to better understand how the plant cell wall responds to the IL before, during, and after pretreatment.