Function and molecular mechanism of a poplar placenta limited MIXTA gene in regulating differentiation of plant epidermal cells.

The placenta in fruits of most plants either desiccate and shrink as the fruits mature or develop further to form the fleshy tissues. In poplars, placental epidermal cells protrude collectively to produce catkin fibers. In this study, three carpel limited MIXTA genes, PdeMIXTA02, PdeMIXTA03, PdeMIXTA04, were find to specifically expressed in carpel immediately after pollination. Heterologous expression of the three genes in Arabidopsis demonstrated that PdeMIXTA04 significantly promoted trichomes density and could restore trichomes in the trichomeless mutant. By contrast, such functions were not observed with PdeMIXTA02, PdeMIXTA03. In situ hybridization revealed that PdeMIXTA04 was explicitly expressed in poplar placental epidermal cells. We also confirmed trichome-specific expression of the PdeMIXTA04 promoter. Multiple experimental proofs have confirmed the interaction between PdeMIXTA04, PdeMYC and PdeWD40, indicating PdeMIXTA04 functioned through the MYB-bHLH-WD40 ternary complex. Our work provided distinctive understanding of the molecular mechanism triggering differentiation of poplar catkins.

Single-cell RNA sequencing reveals a high-resolution cell atlas of xylem in Populus.

High-throughput single-cell RNA sequencing (scRNA-seq) has advantages over traditional RNA-seq to explore spatiotemporal information on gene dynamic expressions in heterogenous tissues. We performed Drop-seq, a method for the dropwise sequestration of single cells for sequencing, on protoplasts from the differentiating xylem of Populus alba × Populus glandulosa. The scRNA-seq profiled 9,798 cells, which were grouped into 12 clusters. Through characterization of differentially expressed genes in each cluster and RNA in situ hybridizations, we identified vessel cells, fiber cells, ray parenchyma cells and xylem precursor cells. Diffusion pseudotime analyses revealed the differentiating trajectory of vessels, fiber cells and ray parenchyma cells and indicated a different differentiation process between vessels and fiber cells, and a similar differentiation process between fiber cells and ray parenchyma cells. We identified marker genes for each cell type (cluster) and key candidate regulators during developmental stages of xylem cell differentiation. Our study generates a high-resolution expression atlas of wood formation at the single cell level and provides valuable information on wood formation.

Prior secondary cell wall formation is required for gelatinous layer deposition and posture control in gravi-stimulated aspen.

Cell walls, especially secondary cell walls (SCWs), maintain cell shape and reinforce wood, but their structure and shape can be altered in response to gravity. In hardwood trees, tension wood is formed along the upper side of a bending stem and contains wood fiber cells that have a gelatinous layer (G-layer) inside the SCW. In a previous study, we generated nst/snd quadruple-knockout aspens (Populus tremula × Populus tremuloides), in which SCW formation was impaired in 99% of the wood fiber cells. In the present study, we produced nst/snd triple-knockout aspens, in which a large number of wood fibers had thinner SCWs than the wild type (WT) and some had no SCW. Because SCW layers are always formed prior to G-layer deposition, the nst/snd mutants raise interesting questions of whether the mutants can form G-layers without SCW and whether they can control their postures in response to changes in gravitational direction. The nst/snd mutants and the WT plants showed growth eccentricity and vessel frequency reduction when grown on an incline, but the triple mutants recovered their upright growth only slightly, and the quadruple mutants were unable to maintain their postures. The mutants clearly showed that the G-layers were formed in SCW-containing wood fibers but not in those lacking the SCW. Our results indicate that SCWs are essential for G-layer formation and posture control. Furthermore, each wood fiber cell may be able to recognize its cell wall developmental stage to initiate the formation of the G-layer as a response to gravistimulation.

Poplar acetylome profiling reveals lysine acetylation dynamics in seasonal bud dormancy release.

For perennials in boreal and temperate ecosystems, bud dormancy is crucial for survival in harsh winter. Dormancy is released by prolonged exposure to low temperatures and is followed by reactive growth in the spring. Lysine acetylation (Kac) is one of the major post-translational modifications (PTMs) that are involved in plant response to environmental signals. However, little information is available on the effects of Kac modification on bud dormancy release. Here, we report the dynamics of lysine acetylome in hybrid poplar (Populus tremula × Populus alba) dormant buds. A total of 7,594 acetyl-sites from 3,281 acetyl-proteins were identified, representing a large dataset of lysine acetylome in plants. Of them, 229 proteins were differentially acetylated during bud dormancy release and were mainly involved in the primary metabolic pathways. Site-directed mutagenesis enzymatic assays showed that Kac strongly modified the activities of two key enzymes of primary metabolism, pyruvate dehydrogenase (PDH) and isocitrate dehydrogenase (IDH). We thus propose that Kac of enzymes could be an important strategy for reconfiguration of metabolic processes during bud dormancy release. In all, our results reveal the importance of Kac in bud dormancy release and provide a new perspective to understand the molecular mechanisms of seasonal growth of trees.

Mild Acetylation and Solubilization of Ground Whole Plant Cell Walls in EmimAc: A Method for Solution-State NMR in DMSO-d6.

Lignocellulosic biomass is mainly composed of polysaccharides and lignin. The complexity and diversity of the plant cell wall polymers makes it difficult to isolate the components in pure form for characterization. Many current approaches to analyzing the structure of lignocellulose, which involve sequential extraction and characterization of the resulting fractions, are time-consuming and labor-intensive. The present study describes a new and facile system for rationally derivatizing and dissolving coarsely ground plant cell wall materials. Using ionic liquids (EmimAc) and dichloroacetyl chloride as a solvent/reagent produced mildly acetylated whole cell walls without significant degradation. The acetylated products were soluble in DMSO-d6 from which they can be characterized by solution-state two-dimensional nuclear magnetic resonance (2D NMR) spectrometry. A distinct advantage of the procedure is that it realizes the dissolution of whole lignocellulosic materials without requiring harsh ball milling, thereby allowing the acquisition of high-resolution 2D NMR spectra to revealing structural details of the main components (lignin and polysaccharides). The method is therefore beneficial to understanding the composition and structure of biomass aimed at its improved utilization.

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.

Genome-wide identification, expression and functional analysis of Populus xylogen-like genes.

As an extracellular arabinogalactan protein (AGP) containing a non-specific lipid transfer protein (nsLTP) domain, xylogen mediates the local intercellular communication required for tracheary element (TE) differentiation in Zinnia cell culture. Although XYLP (xylogen-like protein) gene families have been reported in Arabidopsis and rice, no comprehensive analysis has been performed in woody plants. In this work, 31 XYLP genes in five phylogenetic groups were identified from Populus trichocarpa genome and a comprehensive bioinformatic analysis including gene and protein structures, chromosomal locations and duplication events were conducted. In-silico data and qRT-PCR results indicated that PtXYLP1 is predominantly expressed in poplar apex, young leaves and roots, while PtXYLP2 is uniformly expressed across a variety of tissues with a low abundance. Analysis on PtXYLP1pro:GUS and PtXYLP2pro:GUS in Arabidopsis revealed their differential expression patterns during seed germination and specific inductions by exogenously applied phytohormones including auxin, cytokinin and GA. When overexpressed in Arabidopsis, PtXYLP1 but not PtXYLP2 resulted in cotyledons with defective venation patterns and interrupted secondary (2°) vein loops, which phenotype was underpinned by the down-regulation of genes indispensably required by embryonic venation development at procambium and/or vessel level.

A novel synthetic-genetic-array-based yeast one-hybrid system for high discovery rate and short processing time.

Eukaryotic gene expression is often tightly regulated by interactions between transcription factors (TFs) and their DNA cis targets. Yeast one-hybrid (Y1H) is one of the most extensively used methods to discover these interactions. We developed a high-throughput meiosis-directed yeast one-hybrid system using the Magic Markers of the synthetic genetic array analysis. The system has a transcription factor-DNA interaction discovery rate twice as high as the conventional diploid-mating approach and a processing time nearly one-tenth of the haploid-transformation method. The system also offers the highest accuracy in identifying TF-DNA interactions that can be authenticated in vivo by chromatin immunoprecipitation. With these unique features, this meiosis-directed Y1H system is particularly suited for constructing novel and comprehensive genome-scale gene regulatory networks for various organisms.

An AP2/ERF transcription factor ERF139 coordinates xylem cell expansion and secondary cell wall deposition.

Differentiation of xylem elements involves cell expansion, secondary cell wall (SCW) deposition and programmed cell death. Transitions between these phases require strict spatiotemporal control. The function of Populus ERF139 (Potri.013G101100) in xylem differentiation was characterized in transgenic overexpression and dominant repressor lines of ERF139 in hybrid aspen (Populus tremula × tremuloides). Xylem properties, SCW chemistry and downstream targets were analyzed in both types of transgenic trees using microscopy techniques, Fourier transform-infrared spectroscopy, pyrolysis-GC/MS, wet chemistry methods and RNA sequencing. Opposite phenotypes were observed in the secondary xylem vessel sizes and SCW chemistry in the two different types of transgenic trees, supporting the function of ERF139 in suppressing the radial expansion of vessel elements and stimulating accumulation of guaiacyl-type lignin and possibly also xylan. Comparative transcriptomics identified genes related to SCW biosynthesis (LAC5, LBD15, MYB86) and salt and drought stress-responsive genes (ANAC002, ABA1) as potential direct targets of ERF139. The phenotypes of the transgenic trees and the stem expression profiles of ERF139 potential target genes support the role of ERF139 as a transcriptional regulator of xylem cell expansion and SCW formation, possibly in response to osmotic changes of the cells.

Phosphorylation of LTF1, an MYB Transcription Factor in Populus, Acts as a Sensory Switch Regulating Lignin Biosynthesis in Wood Cells.

Lignin is specifically deposited in plant secondary cell walls, and initiation of lignin biosynthesis is regulated by a variety of developmental and environmental signals. However, the mechanisms governing the regulation of lignin biosynthesis remain to be elucidated. In this study, we identified a lignin biosynthesis-associated transcription factor (LTF) from Populus, LTF1, which binds the promoter of a key lignin biosynthetic gene encoding 4-coumarate-CoA ligase (4CL). We showed that LTF1 in its unphosphorylated state functions as a regulator restraining lignin biosynthesis. When LTF1 becomes phosphorylated by PdMPK6 in response to external stimuli such as wounding, it undergoes degradation through a proteasome pathway, resulting in activation of lignification. Expression of a phosphorylation-null mutant version of LTF1 led to stable protein accumulation and persistent attenuation of lignification in wood cells. Taken together, our study reveals a mechanism whereby LTF1 phosphorylation acts as a sensory switch to regulate lignin biosynthesis in response to environmental stimuli. The discovery of novel modulators and mechanisms modifying lignin biosynthesis has important implications for improving the utilization of cell-wall biomass.

Abscisic acid promotes root system development in birch tissue culture: a comparison to aspen culture and conventional rooting-related growth regulators.

The research aim was to assess the effects of the plant hormone abscisic acid (ABA) and the growth regulator paclobutrazol (PBZ) on root system development during the in vitro culture of different birch and aspen genotypes. The studied genotypes involved two aspen (Populus tremula and Populus tremuloides × P. tremula) and two silver birch (Betula pendula) trees, with one of the birches characterized by its inability to root in vitro. For experiments, apical shoot segments were cultured on nutrient medium enriched with either ABA or PBZ. Additionally, the analysis of the endogenous hormones in shoots developed on hormone-free medium was conducted by high-performance liquid chromatography. The endogenous concentration of auxin indole-3-acetic acid was much higher in the aspens than that in the birches, while the highest concentration of ABA was found in the root-forming birch. The culturing of this birch genotype on medium enriched with ABA resulted in an increased root length and a higher number of lateral roots without any negative effect on either shoot growth or adventitious root (AR) formation, although these two processes were largely inhibited by ABA in the aspens. Meanwhile, PBZ promoted AR formation in both aspen and birch cultures but impaired secondary root formation and shoot growth in birches. These results suggest the use of ABA for the in vitro rooting of birches and PBZ for the rooting of aspens.

Autophagy counteracts instantaneous cell death during seasonal senescence of the fine roots and leaves in Populus trichocarpa.

BACKGROUND: Senescence, despite its destructive character, is a process that is precisely-regulated. The control of senescence is required to achieve remobilization of resources, a principle aspect of senescence. Remobilization allows plants to recapture valuable resources that would otherwise be lost to the environment with the senescing organ. Autophagy is one of the critical processes that is switched on during senescence. This evolutionarily conserved process plays dual, antagonistic roles. On the one hand, it counteracts instantaneous cell death and allows the process of remobilization to be set in motion, while on the other hand, it participates in the degradation of cellular components. Autophagy has been demonstrated to occur in many plant species during the senescence of leaves and flower petals. Little is known, however, about the senescence process in other ephemeral organs, such as fine roots, whose lifespan is also relatively short. We hypothesized that, like the case of seasonal leaf senescence, autophagy also plays a role in the senescence of fine roots, and that both processes are synchronized in their timing. RESULTS: We evaluated which morphological and cytological symptoms are universal or unique in the senescence of fine roots and leaves. The results of our study confirmed that autophagy plays a key role in the senescence of fine roots, and is associated also with the process of cellular components degradation. In both organs, structures related to autophagy were observed, such as autophagic bodies and autophagosomes. The role of autophagy in the senescence of these plant organs was further confirmed by an analysis of ATG gene expression and protein detection. CONCLUSIONS: The present study is the first one to examine molecular mechanisms associated with the senescence of fine roots, and provide evidence that can be used to determine whether senescence of fine roots can be treated as another example of developmentally programmed cell death (dPCD). Our results indicate that there is a strong similarity between the senescence of fine roots and other ephemeral organs, suggesting that this process occurs by the same autophagy-related mechanisms in all plant ephemeral organs.

Preliminary study of Cell Wall Structure and its Mechanical Properties of C3H and HCT RNAi Transgenic Poplar Sapling.

This research focused on the cell wall structure and its mechanical properties of down-regulated Coumaroyl shikimate 3-hydroxylase (C3H) transgenic poplar and down-regulated hydroxycinnamoyl CoA: shikimate hydroxycinnamoyl transferase (HCT) transgenic poplar (Populus alba × P. glandulosa cv '84 k'). The wood samples with respect to microstructure, the longitudinal elastic modulus (MOE) and hardness of wood fiber secondary cell wall were investigated. The results show that the lignin contents in the two transgenic poplar woods were lower than non-modified wood. The C3H transgenic poplar and HCT transgenic poplar have more than 18.5% and 16.1% cellulose crystalline regions than non-modified poplar respectively. The diameter of the fiber cell and the vessel element of transgenic poplars are smaller. Double radial vessel cell wall thicknesses of both transgenic poplars were smaller than non-modified poplar. Cell wall ratios for the transgenic poplar were higher than non-modified poplar and cell wall density was significantly lower in both C3H and HCT transgenic poplar. The cell wall MOEs of C3H and HCT transgenic poplar was 5.8% and 7.0% higher than non-modified poplar. HCT can be more effective than C3H to modify the trees by considerably increasing mechanical properties of the cell wall.

Imaging the dynamic deposition of cell wall polymer in xylem and phloem in Populus × euramericana.

MAIN CONCLUSION: Both G units and S units deposited in the whole lignification process of xylem fiber. The topochemical variations in newly formed xylem and phloem of Populus × euramericana were investigated by combined microscopic techniques. During xylem formation, earlier cell wall deposition in vessel and afterwards in the neighboring fiber was observed in situ. Raman images in xylem fiber emphasized that cell wall deposition was an ordered process which lignification started in cell corner following carbohydrates deposition. Higher deposition speed of carbohydrates was revealed at the beginning of the cell wall differentiation, and the syringyl (S) units deposition was more pronounced compared with guaiacyl (G) units at the earlier stage of lignification. The comparative analysis of cell wall composition in phloem fiber indicated that phloem formed earlier than xylem and the distribution of lignin monomers varied significantly with phloem fiber location. Furthermore, an interesting phenomenon was found that the outermost phloem fiber near the periderm displayed a multilayered structure with alternating broad and narrow layer, and the broad lamellae showed higher concentration of carbohydrates and S lignin. The cytological information including cell wall composition and lignin structure of xylem and phloem might be helpful to understand the wood growth progresses and facilitate utilization of woody plants.

Infrared Nanospectroscopy Reveals the Chemical Nature of Pit Membranes in Water-Conducting Cells of the Plant Xylem.

In the xylem of angiosperm plants, microscopic pits through the secondary cell walls connect the water-conducting vessels. Cellulosic meshes originated from primary walls, and middle lamella between adjacent vessels, called the pit membrane, separates one conduit from another. The intricate structure of the nano-sized pores in pit membranes enables the passage of water under negative pressure without hydraulic failure due to obstruction by gas bubbles (i.e. embolism) under normal conditions or mild drought stress. Since the chemical composition of pit membranes affects embolism formation and bubble behavior, we directly measured pit membrane composition in Populus nigra wood. Here, we characterized the chemical composition of cell wall structures by synchrotron infrared nanospectroscopy and atomic force microscopy-infrared nanospectroscopy with high spatial resolution. Characteristic peaks of cellulose, phenolic compounds, and proteins were found in the intervessel pit membranes of P. nigra wood. In addition, the vessel to parenchyma pit membranes and developing cell walls of the vascular cambium showed clear signals of cellulose, proteins, and pectin. We did not find a distinct peak of lignin and other compounds in these structures. Our investigation of the complex chemical composition of intervessel pit membranes furthers our understanding of the flow of water and bubbles between neighboring conduits. The advances presented here pave the way for further label-free studies related to the nanochemistry of plant cell components.

Cellulose Synthase Stoichiometry in Aspen Differs from Arabidopsis and Norway Spruce.

Cellulose is synthesized at the plasma membrane by cellulose synthase complexes (CSCs) containing cellulose synthases (CESAs). Genetic analysis and CESA isoform quantification indicate that cellulose in the secondary cell walls of Arabidopsis (Arabidopsis thaliana) is synthesized by isoforms CESA4, CESA7, and CESA8 in equimolar amounts. Here, we used quantitative proteomics to investigate whether the CSC model based on Arabidopsis secondary cell wall CESA stoichiometry can be applied to the angiosperm tree aspen (Populus tremula) and the gymnosperm tree Norway spruce (Picea abies). In the developing xylem of aspen, the secondary cell wall CESA stoichiometry was 3:2:1 for PtCESA8a/b:PtCESA4:PtCESA7a/b, while in Norway spruce, the stoichiometry was 1:1:1, as observed previously in Arabidopsis. Furthermore, in aspen tension wood, the secondary cell wall CESA stoichiometry changed to 8:3:1 for PtCESA8a/b:PtCESA4:PtCESA7a/b. PtCESA8b represented 73% of the total secondary cell wall CESA pool, and quantitative polymerase chain reaction analysis of CESA transcripts in cryosectioned tension wood revealed increased PtCESA8b expression during the formation of the cellulose-enriched gelatinous layer, while the transcripts of PtCESA4, PtCESA7a/b, and PtCESA8a decreased. A wide-angle x-ray scattering analysis showed that the shift in CESA stoichiometry in tension wood coincided with an increase in crystalline cellulose microfibril diameter, suggesting that the CSC CESA composition influences microfibril properties. The aspen CESA stoichiometry results raise the possibility of alternative CSC models and suggest that homomeric PtCESA8b complexes are responsible for cellulose biosynthesis in the gelatinous layer in tension wood.

Storage lipid accumulation is controlled by photoperiodic signal acting via regulators of growth cessation and dormancy in hybrid aspen.

The signalling pathways that control seasonal modulation of carbon metabolism in perennial plants are poorly understood. Using genetic, metabolic and natural variation approaches, we identify factors mediating photoperiodic control of storage lipid accumulation in the model tree hybrid aspen (Populus tremula × tremuloides). We characterized lipid accumulation in transgenic hybrid aspen with impaired photoperiodic and hormonal responses. Genome-wide association mapping was performed in Swedish aspen (P. tremula) genotypes to determine genetic loci associated with genotype variation in lipid content. Our data show that the storage lipid triacylglycerol (TAG) accumulates in cambial meristem and pith rays of aspen in response to photoperiodic signal controlling growth cessation and dormancy induction. We show that photoperiodic control of TAG accumulation is mediated by the FLOWERING LOCUS T/CONSTANS module, which also controls the induction of growth cessation. Hormonal and chromatin remodelling pathways also contribute to TAG accumulation by photoperiodic signal. Natural variation exists in lipid accumulation that is controlled by input from multiple loci. Our data shed light on how the control of storage metabolism is temporally coordinated with growth cessation and dormancy by photoperiodic signal, and reveals that storage lipid accumulation between seeds and perennating organs of trees may involve distinct regulatory circuits.

Can sugar metabolism in the cambial region explain the water deficit tolerance in poplar?

Drought dramatically affects wood production by adversely impacting cambial cells and their derivatives. Photosynthesis and assimilate transport are also affected by drought conditions. Two poplar genotypes, Populus deltoides 'Dvina' and Populus alba 'Marte', demonstrated contrasting growth performance and water-carbon balance strategies; a mechanistic understanding of the water deficit response was provided by these poplar species. 'Marte' was found to be more anisohydric than 'Dvina'. This characteristic was associated with the capacity to reallocate carbohydrates during water deficits. In contrast, 'Dvina' displayed more conservative water management; carbohydrates were preferably stored or used for cellulose production rather than to achieve an osmotic balance between the phloem and the xylem. Data confirmed that the more 'risk-taking' characteristic of 'Marte' allowed a rapid recovery following water deficit and was connected to a different carbohydrate metabolism.

Identification of recombination events in outbred species with next-generation sequencing data.

BACKGROUND: Meiotic recombination events include crossovers and non-crossovers or gene conversions. Although the rate of crossovers is often used for genetic mapping, the gene conversion events are not well studied especially in outbred species, which could produce distorted markers and thus affect the precision of genetic maps. RESULTS: We proposed a strategy for identifying gene conversion events in Populus with the next-generation sequencing (NGS) data from the two parents and their progeny in an F1 hybrid population. The strategy first involved phasing the heterozygous SNPs of the parents to obtain the parental haplotype blocks by NGS analytical tools, permitting to identify the parental gene conversion events with progeny genotypes. By incorporating available genetic linkage maps, longer haplotype blocks each corresponding to a chromosome can be created, not only allowing to detect crossover events but also possibly to locate a crossover in a small region. Our analysis revealed that gene conversions are more abundant than crossovers in Populus, with a higher probability to generate distorted markers in the regions involved than in the other regions on genome. The analytical procedures were implemented with Perl scripts as a freely available package, findGCO at https://github.com/tongchf/findGCO . CONCLUSIONS: The novel strategy and the new developed Perl package permit to identify gene conversion events with the next-generation sequencing technology in a hybrid population of outbred species. The new method revealed that in a genetic mapping population some distorted genetic markers are possibly due to the gene conversion events.

Photoperiodic control of seasonal growth is mediated by ABA acting on cell-cell communication.

In temperate and boreal ecosystems, seasonal cycles of growth and dormancy allow perennial plants to adapt to winter conditions. We show, in hybrid aspen trees, that photoperiodic regulation of dormancy is mechanistically distinct from autumnal growth cessation. Dormancy sets in when symplastic intercellular communication through plasmodesmata is blocked by a process dependent on the phytohormone abscisic acid. The communication blockage prevents growth-promoting signals from accessing the meristem. Thus, precocious growth is disallowed during dormancy. The dormant period, which supports robust survival of the aspen tree in winter, is due to loss of access to growth-promoting signals.