Structural Imaging of Native Cryo-Preserved Secondary Cell Walls Reveals the Presence of Macrofibrils and Their Formation Requires Normal Cellulose, Lignin and Xylan Biosynthesis

The woody secondary cell walls of plants are the largest repository of renewable carbon biopolymers on the planet. These walls are made principally from cellulose and hemicelluloses and are impregnated with lignin. Despite their importance as the main load bearing structure for plant growth, as well as their industrial importance as both a material and energy source, the precise arrangement of these constituents within the cell wall is not yet fully understood. We have adapted low temperature scanning electron microscopy (cryo-SEM) for imaging the nanoscale architecture of angiosperm and gymnosperm cell walls in their native hydrated state. Our work confirms that cell wall macrofibrils, cylindrical structures with a diameter exceeding 10 nm, are a common feature of the native hardwood and softwood samples. We have observed these same structures in Arabidopsis thaliana secondary cell walls, enabling macrofibrils to be compared between mutant lines that are perturbed in cellulose, hemicellulose, and lignin formation. Our analysis indicates that the macrofibrils in Arabidopsis cell walls are dependent upon the proper biosynthesis, or composed, of cellulose, xylan, and lignin. This study establishes that cryo-SEM is a useful additional approach for investigating the native nanoscale architecture and composition of hardwood and softwood secondary cell walls and demonstrates the applicability of Arabidopsis genetic resources to relate fibril structure with wall composition and biosynthesis.Peer reviewe

Structural differences of cell walls in earlywood and latewood of Pinus sylvestris and their contribution to biomass recalcitrance

Scots pine (Pinus sylvestris L.) is an evergreen coniferous tree with wide distribution and good growth performance in a range of habitats. Therefore, wood from P. sylvestris is produced in many managed forests and is frequently used in industry. Despite the importance of pine wood, we still do not fully understand its molecular structure what limits improvements in its processing. One of the basic features leading to variation in wood properties is the presence of earlywood and latewood which form annual growth rings. Here, we characterise biochemical traits that differentiate cell walls of earlywood and latewood in Scots pine. We discover that latewood is less recalcitrant to enzymatic digestion, with galactoglucomannan showing particularly pronounced difference in accessibility. Interestingly, characterisation of lignin reveals a higher proportion of coniferaldehydes in pine latewood and suggests the presence of a different linkage landscape in this wood type. With complementary analysis of wood polysaccharides this enabled us to propose the first detailed molecular model of earlywood and latewood and to conclude that the variation in lignin structure is likely the main determinant of differences in recalcitrance observed between the two wood types in pine. Our discoveries lay the foundation for improvements in industrial processes that use pine wood since we show clear pathways for increasing the efficiency of enzymatic processing of this renewable material. Our work will help guide future breeding of pine trees with desired timber properties and can help link molecular structure of softwood cell walls to function of the different types of xylem in conifers

Microscopy and chemical analyses reveal flavone-based woolly fibres extrude from micron-sized holes in glandular trichomes of Dionysia tapetodes.

BackgroundDionysia tapetodes, a small cushion-forming mountainous evergreen in the Primulaceae, possesses a vast surface-covering of long silky fibres forming the characteristic "woolly" farina. This contrasts with some related Primula which instead form a fine powder. Farina is formed by specialized cellular factories, a type of glandular trichome, but the precise composition of the fibres and how it exits the cell is poorly understood. Here, using a combination of cell biology (electron and light microscopy) and analytical chemical techniques, we present the principal chemical components of the wool and its mechanism of exit from the glandular trichome.ResultsWe show the woolly farina consists of micron-diameter fibres formed from a mixture of flavone and substituted flavone derivatives. This contrasts with the powdery farina, consisting almost entirely of flavone. The woolly farina in D. tapetodes is extruded through specific sites at the surface of the trichome's glandular head cell, characterised by a small complete gap in the plasma membrane, cell wall and cuticle and forming a tight seal between the fibre and hole. The data is consistent with formation and thread elongation occurring from within the cell.ConclusionsOur results suggest the composition of the D. tapetodes farina dictates its formation as wool rather than powder, consistent with a model of thread integrity relying on intermolecular H-bonding. Glandular trichomes produce multiple wool fibres by concentrating and maintaining their extrusion at specific sites at the cell cortex of the head cell. As the wool is extensive across the plant, there may be associated selection pressures attributed to living at high altitudes

Specific antibody responses against membrane proteins of erythrocytes infected by Plasmodium falciparum of individuals briefly exposed to malaria

International audienceBACKGROUND: Plasmodium falciparum infections could lead to severe malaria, principally in non-immune individuals as children and travellers from countries exempted of malaria. Severe malaria is often associated with the sequestration of P. falciparum-infected erythrocytes in deep micro-vascular beds via interactions between host endothelial receptors and parasite ligands expressed on the surface of the infected erythrocyte. Although, serological responses from individuals living in endemic areas against proteins expressed at surface of the infected erythrocyte have been largely studied, seldom data are available about the specific targets of antibody response from travellers. METHODS: In order to characterize antigens recognized by traveller sera, a comparison of IgG immune response against membrane protein extracts from uninfected and P. falciparum-infected red blood cells (iRBC), using immunoblots, was performed between non exposed individuals (n = 31) and briefly exposed individuals (BEI) (n = 38) to malaria transmission. RESULTS: Immune profile analysis indicated that eight protein bands from iRBC were significantly detected more frequently in the BEI group. Some of these antigenic proteins were identified by an original immuno-proteomic approach. CONCLUSION: Collectively, these data may be useful to characterize the singular serological immune response against a primary malaria infection in individuals briefly exposed to transmission

A root phloem pole cell atlas reveals common transcriptional states in protophloem-adjacent cells

The phloem pole atlas has over 10,000 cells, with an unprecedented resolution of the transcriptional dynamics in phloem development. Despite distinct mature transcriptional states, co-expression networks show common states in protophloem-adjacent cells. Single-cell sequencing has recently allowed the generation of exhaustive root cell atlases. However, some cell types are elusive and remain underrepresented. Here we use a second-generation single-cell approach, where we zoom in on the root transcriptome sorting with specific markers to profile the phloem poles at an unprecedented resolution. Our data highlight the similarities among the developmental trajectories and gene regulatory networks common to protophloem sieve element (PSE)-adjacent lineages in relation to PSE enucleation, a key event in phloem biology. As a signature for early PSE-adjacent lineages, we have identified a set of DNA-binding with one finger (DOF) transcription factors, the PINEAPPLEs (PAPL), that act downstream of PHLOEM EARLY DOF (PEAR) genes and are important to guarantee a proper root nutrition in the transition to autotrophy. Our data provide a holistic view of the phloem poles that act as a functional unit in root development.Peer reviewe

Interactions between callose and cellulose revealed through the analysis of biopolymer mixtures.

The properties of (1,3)-β-glucans (i.e., callose) remain largely unknown despite their importance in plant development and defence. Here we use mixtures of (1,3)-β-glucan and cellulose, in ionic liquid solution and hydrogels, as proxies to understand the physico-mechanical properties of callose. We show that after callose addition the stiffness of cellulose hydrogels is reduced at a greater extent than predicted from the ideal mixing rule (i.e., the weighted average of the individual components' properties). In contrast, yield behaviour after the elastic limit is more ductile in cellulose-callose hydrogels compared with sudden failure in 100% cellulose hydrogels. The viscoelastic behaviour and the diffusion of the ions in mixed ionic liquid solutions strongly indicate interactions between the polymers. Fourier-transform infrared analysis suggests that these interactions impact cellulose organisation in hydrogels and cell walls. We conclude that polymer interactions alter the properties of callose-cellulose mixtures beyond what it is expected by ideal mixing

p73α isoforms drive opposite transcriptional and post-transcriptional regulation of MYCN expression in neuroblastoma cells

MYCN activation, mainly by gene amplification, is one of the most frequent molecular events in neuroblastoma (NB) oncogenesis, and is associated with increased malignancy and decreased neuronal differentiation propensity. The frequency of concomitant loss of heterozygosity at the 1p36.3 locus, which harbours the p53 anti-oncogene homologue TP73, indicates that MYCN and p73 alterations may cooperate in the pathogenesis of NB. We have previously shown that p73 isoforms are deregulated in NB tumours and that TAp73 co-operates synergistically with p53 for apoptosis of NB cells, whereas ΔNp73 activates the expression of neuronal differentiation genes such as BTG2. Herein, using both ectopic expression and RNA interference-mediated silencing of p73 in MYCN amplified NB cells, we show that p73α isoforms inhibit MYCN expression at both transcript and protein levels, in spite of transactivator effects on MYCN promoter. To explain this paradox, we found that TAp73α exerts negative post-transcriptional effects leading to reduced MYCN mRNA stability. RNA immunoprecipitation experiments suggest that this dominant inhibitory post-transcriptional effect could be due to an interaction between the p73 protein and MYCN mRNA, a hypothesis also raised for the regulation of certain genes by the p53 protein

Ectopic callose deposition into woody biomass modulates the nano-architecture of macrofibrils

Plant biomass plays an increasingly important role in the circular bioeconomy, replacing non-renewable fossil resources. Genetic engineering of this lignocellulosic biomass could benefit biorefinery transformation chains by lowering economic and technological barriers to industrial processing. However, previous efforts have mostly targeted the major constituents of woody biomass: cellulose, hemicellulose and lignin. Here we report the engineering of wood structure through the introduction of callose, a polysaccharide novel to most secondary cell walls. Our multiscale analysis of genetically engineered poplar trees shows that callose deposition modulates cell wall porosity, water and lignin contents and increases the lignin-cellulose distance, ultimately resulting in substantially decreased biomass recalcitrance. We provide a model of the wood cell wall nano-architecture engineered to accommodate the hydrated callose inclusions. Ectopic polymer introduction into biomass manifests in new physico-chemical properties and offers new avenues when considering lignocellulose engineering.Bourdon et al. demonstrate the possibility to ectopically synthesize callose, a polymer restricted to primary cell walls, into Arabidopsis and aspen secondary cell walls to manipulate their ultrastructure and ultimately reduce their recalcitrance

Unravelling Chemical Composition of Agave Spines: News from Agave fourcroydes Lem.

Spines are key plant modifications developed to deal against herbivores; however, its physical structure and chemical composition have been little explored in plant species. Here, we took advantage of high-throughput chromatography to characterize chemical composition of Agave fourcroydes Lem. spines, a species traditionally used for fiber extraction. Analyses of structural carbohydrate showed that spines have lower cellulose content than leaf fibers (52 and 72%, respectively) but contain more than 2-fold the hemicellulose and 1.5-fold pectin. Xylose and galacturonic acid were enriched in spines compared to fibers. The total lignin content in spines was 1.5-fold higher than those found in fibers, with elevated levels of syringyl (S) and guaiacyl (G) subunits but similar S/G ratios within tissues. Metabolomic profiling based on accurate mass spectrometry revealed the presence of phenolic compounds including quercetin, kaempferol, (+)-catechin, and (-)-epicatechin in A. fourcroydes spines, which were also detected in situ in spines tissues and could be implicated in the color of these plants' structures. Abundance of (+)-catechins could also explain proanthocyanidins found in spines. Agave spines may become a plant model to obtain more insights about cellulose and lignin interactions and condensed tannin deposition, which is valuable knowledge for the bioenergy industry and development of naturally dyed fibers, respectively

L’endoréduplication dans le développement du fruit de tomate : de la structure à la croissance cellulaire

Le développement du fruit de tomate s’accompagne d’un phénomène d’endopolyploïdisation(amplification de l’ADN en l'absence de mitose) associé à la croissance cellulaire. Au stade vert mature huit niveaux de ploïdie sont présents (2C à 256C) dans le péricarpe.Une première partie du travail a porté sur l’étude de la distribution spatiale des niveaux de ploïdie dans ce tissu. Cet objectif a nécessité la mise au point d’une méthode originale de détermination de la ploïdie in situ reposant sur la technique de BAC-FISH. Nous avons montré que les cellules les plus polyploïdes se situent dans les assises internes du péricarpe, et qu’elles sont aussi les plus grandes. Ces cellules semblent déjà formées au moment de l’anthèse. Cette cartographie de la ploïdie associée à une analyse de la taille cellulaire a également montré que la taille finale des cellules ne dépend pas uniquement de leur niveau de ploïdie mais également de leur position dans le péricarpe. Enfin, nos résultats suggèrent que l’endopolyploïdisation précède la croissance cellulaire.Dans une deuxième partie du travail, nous avons étudié la structure des noyaux en microscopie à fluorescence et électronique. L’endopolyploïdisation affecte profondément la taille et la forme des noyaux, qui acquièrent un volume important et une forme complexe avec de profondes invaginations. La taille du nucléole augmente avec celle du noyau, ce qui suggère une activité de transcription accrue. De plus, la présence de nombreuses mitochondries à proximité des noyaux polyploïdes suggère une forte activité métabolique en lien avec l’endopolyploïdisation. L’utilisation de la méthode BAC-FISH a permis également de montrer que la polyploïdie se faisait par endoreduplication avec la formation de chromosomes polytènes.Dans une troisième partie nous avons cherché, en criblant une banque de mutants Micro-Tom, à identifier des lignées affectées dans l’endoreduplication afin d’étudier l’impact de ce phénomène sur la vitesse de croissance du fruit. Nous avons caractérisé plusieurs familles dont les niveaux moyens de ploïdie variaient par rapport à la lignée de référence. Une de ces familles présente un phénotype stable au cours de deux générations, avec une augmentation d’au moins 30 % de la ploïdie moyenne et une augmentation de la taille des cellules du péricarpe. Cependant cette famille présentant aussi un développement relativement parthénocarpique de ses fruits, sa caractérisation n’a pas pu être poursuivie dans le cadre de ce travail.Tomato fruit development includes massive endopolyploidisation events (DNA duplication inthe absence of mitoses) within pericarp cells, in which 8 DNA levels from 2 C to 256 C are detected atmature green stage.The first part of this work dealt with the study of the spatial distribution of ploidy levels inpericarp. To achieve this purpose, a new method for in situ ploidy assessment was set up using aBAC-FISH protocol. The main results are 1/ the most polyploid cells are located in central mesocarpcell layers; 2/ the most polyploid cells are also the largest cells; 3/ these cells are likely to be alreadypresent in ovary at anthesis. Ploidy mapping has also shown that the final cell size does not dependonly on ploidy level but also on cell location in pericarp, and that endopolyploidization is likely set up intissues before cell expansion.The structure of the polyploid nucleus was studied by using fluorescence microscopy andelectron microscopy. Endopolyploidization profoundly modifies the size and shape of nuclei, whichbecome much larger and acquire a complex shape with deep invaginations. Nucleolus size increases,which is likely related to transcriptional increase. Moreover, the presence of numerous mitochondria inthe close vicinity of the nuclear membrane reinforces the hypothesis of increased nuclear andmetabolic activity in polyploid cells. The BAC-FISH in situ method for ploidy assessment also revealedthat endopolyploidization proceeded through polyteny.In the last part of this work, we screened a tomato Micro-Tom tilling bank for mutants affectedin endopolyploidization. The aim was to use tomato lines with distinct ploidy levels to check theinfluence of ploidy on fruit growth rate. Several mutant families were identified with moderatelyincreased ploidy levels. One of these families exhibited transmissible phenotype through 2generations, with ploidy increased by ca. 30 % and increased pericarp cell size. As these mutants hadalso a strongly pronounced parthenocarpic phenotype, their characterization could not be furtheradvanced in the frame of this work