Architecture and permeability of post-cytokinesis plasmodesmata lacking cytoplasmic sleeves

This work was supported by the grants by the Region Aquitaine (to E.M.B) and PEPS (Initial Support for Exploratory Projects to E.M.B) and National Agency for Research (Grant ANR-14-CE19-0006-01 to E.M.B).Plasmodesmata are remarkable cellular machines responsible for the controlled exchange of proteins, small RNAs and signalling molecules between cells. They are lined by the plasma membrane (PM), contain a strand of tubular endoplasmic reticulum (ER), and the space between these two membranes is thought to control plasmodesmata permeability. Here, we have reconstructed plasmodesmata three-dimensional (3D) ultrastructure with an unprecedented level of 3D information using electron tomography. We show that within plasmodesmata, ER-PM contact sites undergo substantial remodelling events during cell differentiation. Instead of being open pores, post-cytokinesis plasmodesmata present such intimate ER-PM contact along the entire length of the pores that no intermembrane gap is visible. Later on, during cell expansion, the plasmodesmata pore widens and the two membranes separate, leaving a cytosolic sleeve spanned by tethers whose presence correlates with the appearance of the intermembrane gap. Surprisingly, the post-cytokinesis plasmodesmata allow diffusion of macromolecules despite the apparent lack of an open cytoplasmic sleeve, forcing the reassessment of the mechanisms that control plant cell-cell communication.PostprintPeer reviewe

High intraspecific variation of the cell surface physico-chemical and bioadhesion properties in Brettanomyces bruxellensis

Brettanomyces bruxellensis is the most damaging spoilage yeast in the wine industry because of its negative impact on the wine organoleptic qualities. The strain persistence in cellars over several years associated with recurrent wine contamination suggest specific properties to persist and survive in the environment through bioadhesion phenomena. In this work, the physico-chemical surface properties, morphology and ability to adhere to stainless steel were studied both on synthetic medium and on wine. More than 50 strains representative of the genetic diversity of the species were considered. Microscopy techniques made it possible to highlight a high morphological diversity of the cells with the presence of pseudohyphae forms for some genetic groups. Analysis of the physico-chemical properties of the cell surface reveals contrasting behaviors: most of the strains display a negative surface charge and hydrophilic behavior while the Beer 1 genetic group has a hydrophobic behavior. All strains showed bioadhesion abilities on stainless steel after only 3 h with differences in the concentration of bioadhered cells ranging from 2.2 × 102 cell/cm2 to 7.6 × 106 cell/cm2. Finally, our results show high variability of the bioadhesion properties, the first step in the biofilm formation, according to the genetic group with the most marked bioadhesion capacity for the beer group. © 2023 The Author

Proteomic Analysis of Lipid Droplets from Arabidopsis Aging Leaves Brings New Insight into Their Biogenesis and Functions

Lipid droplets (LDs) are cell compartments specialized for oil storage. Although their role and biogenesis are relatively well documented in seeds, little is known about their composition, structure and function in senescing leaves where they also accumulate. Here, we used a label free quantitative mass spectrometry approach to define the LD proteome of aging Arabidopsis leaves. We found that its composition is highly different from that of seed/cotyledon and identified 28 proteins including 9 enzymes of the secondary metabolism pathways involved in plant defense response. With the exception of the TRIGALACTOSYLDIACYLGLYCEROL2 protein, we did not identify enzymes implicated in lipid metabolism, suggesting that growth of leaf LDs does not occur by local lipid synthesis but rather through contact sites with the endoplasmic reticulum (ER) or other membranes. The two most abundant proteins of the leaf LDs are the CALEOSIN3 and the SMALL RUBBER PARTICLE1 (AtSRP1); both proteins have structural functions and participate in plant response to stress. CALEOSIN3 and AtSRP1 are part of larger protein families, yet no other members were enriched in the LD proteome suggesting a specific role of both proteins in aging leaves. We thus examined the function of AtSRP1 at this developmental stage and found that AtSRP1 modulates the expression of CALEOSIN3 in aging leaves. Furthermore, AtSRP1 overexpression induces the accumulation of triacylglycerol with an unusual composition compared to wild-type. We demonstrate that, although AtSRP1 expression is naturally increased in wild type senescing leaves, its overexpression in senescent transgenic lines induces an over-accumulation of LDs organized in clusters at restricted sites of the ER. Conversely, atsrp1 knock-down mutants displayed fewer but larger LDs. Together our results reveal that the abundancy of AtSRP1 regulates the neo-formation of LDs during senescence. Using electron tomography, we further provide evidence that LDs in leaves share tenuous physical continuity as well as numerous contact sites with the ER membrane. Thus, our data suggest that leaf LDs are functionally distinct from seed LDs and that their biogenesis is strictly controlled by AtSRP1 at restricted sites of the ER

Etudes génétiques de la symbiose Medicago truncatula-Sinorhizobium melitoti (utilisation d'approches de génétique inverse pour l'étude de la fonction biologique d'ENOD40)

Les légumineuses sont capables de s associer avec des bactéries du sol pour former un nouvel organe: la nodosité. Le modèle utilisé au laboratoire pour l étude de cette symbiose correspond au couple Medicago truncatula-Sinorhizobium meliloti. Afin de tenter de mieux comprendre la fonction du gène ENOD40, des approches par silencing ont été initiées. Des lignées transgéniques de M. truncatula, qui expriment une construction ARNi (ARN interference) dirigée contre ENOD40, ont été produites. Les analyses phénotypiques et moléculaires de ces lignées montrent que l induction de ce gène n est pas requise pour cette association symbiotique. De plus, au cours de ce travail, un deuxième gène ENOD40, MtENOD40-2, a été identifié chez M. truncatula. MtENOD40-2 se différencie de MtENOD40-1 par son profil de transcription mais également par la séquence du peptide I putativement codé par la majorité des gènes ENOD40 identifiés. En parallèle, afin de découvrir de nouveaux mutants de nodulation et les gènes correspondants, le criblage de deux collections de lignées d insertion, soit ADN-T soit Tnt1, de M. truncatula a été initié. Sur 271 lignées ADN-T criblées, quatre présentent des phénotypes symbiotiques qui ne co-ségrègent pas avec l étiquette mutagène ; tandis que deux mutants symbiotiques étiquetés ont été identifiés parmi 200 lignées Tnt1. La production ainsi que le criblage de nouvelles lignées Tnt1 semblent constituer un outil plus efficace pour la découverte, chez M. truncatula, de nouveaux gènes symbiotiques, par génétique directe, et de nouveaux mutants d intérêt, par génétique inverse.The symbiotic interaction between leguminous plants and soil bacteria leads to the formation of a new organ : the nodule. The molecular mechanisms controlling this interaction are not yet well understood. The interaction between Medicago truncatula and Sinorhizobium meliloti represents an interesting model to study this symbiotic interaction. We have used a silencing approach with RNAi to investigate the function of the ENOD40 gene that is induced during the symbiotic interaction and encodes a short ORFs-containing RNA. Because of the lack of an appropriated viral vector for Virus-induced gene silencing (VIGS) in M. truncatula, transgenic lines expressing an RNAi construct directed against ENOD40 were produced. The phenotype and the molecular analysis of these transgenic lines, analyzed under three different growth conditions indicated that the ENOD40 induction is not required for the symbiotic interaction. Moreover, a second MtENOD40 gene, MtENOD40-2, was discovered during this study. These MTENOD40 genes differ by their transcription patterns and their putative peptide I sequences. In addition, in order to discover new nodulation mutants and the corresponding genes, we initiated the screening of two insertion mutant collections (T-DNA or Tnt1). Four untagged and two tagged symbiotic mutants were isolated using respectively 271 T-DNA lines and 200 Tnt1 lines. Thus, as a result of the multicopy nature of the Tnt1 element in the Tnt1 lines, screening of these lines seems to be more efficient for the discovery of new symbiotic mutants in M. truncatula by forward and reverse genetic approaches.ORSAY-PARIS 11-BU Sciences (914712101) / SudocSudocFranceF

A mechanically sensitive cell layer regulates the physical properties of the Arabidopsis seed coat

International audienceEndogenous mechanical stresses regulate plant growth and development. Tensile stress in epidermal cells affects microtubule reorientation and anisotropic cell wall deposition, and mechanical stimulus at the meristem regulates trafficking and polar localization of auxin transporters. However, the mechanical regulation of other plant growth regulators has not been demonstrated. Here we propose that during seed growth, mechanical stress exerted by the expanding embryo and endosperm is perceived by a specific mechanosensitive cell layer in the seed coat. We show that the adaxial epidermis of the outer integument thickens its cell wall in a mechanosensitive fashion, demonstrates microtubule dynamics consistent with mechanical stress perception and shows mechanosensitive expression of ELA1, a regulator of seed size and gibberellic acid (GA) metabolism. By exploiting physical and genetic compartmentalization, and combining genetic and surgical techniques, we propose a mechanistic link between mechanical stress and GA accumulation that regulates seed development

Electron tomography to study the three-dimensional structure of Plasmodesmata in plant tissues–from high pressure freezing preparation to ultrathin section collection

Plasmodesmata (PD) are nanometric (similar to 20 nm wide) membrane lined pores encased in the cell walls of the adjacent plant cells. They allow the cells to exchange all types of molecules ranging from nutrients like sugar, hormones, to RNAs and various proteins. Unfortunately, they are also hijacked by phyto-viruses, enabling them to spread from cell-to-cell and then systematically throughout the whole plant. Their central position in plant biology makes it crucial to understand their physiology and especially link their function to their structure. Over the past 50 years, electron microscopists have observed them and attempted to ultrastructurally characterize them. They laid the foundation of what is known about these pores (Tilney et al., 1991; Ding et al., 1992; Oparka and Roberts, 2001; Nicolas et al., 2017a). Despite the explosion of three-dimensional electron microscopy (3D-EM), PD ultrastructure remained recalcitrant to such technique. The first technical difficulty is to process them in such a way where they are as close to their native state as possible. Secondly, plant samples reveal themselves as being difficult to process due to the poor staining/fixating reagents penetration rates, their increased size, their high water content and the presence of an acidic vacuole. On top of this, their very unique position in the cell wall and their nanometric size make them difficult to conveniently stain in order to see the inner-workings of these pores. Here we describe in detail the protocol used in Nicolas et al. (2017b) to image PD in fine detail and produce high-resolution tomograms

AP-1 is required for the maintenance of apico-basal polarity in the C. elegans intestine.

International audienceEpithelial tubes perform functions that are essential for the survival of multicellular organisms. Understanding how their polarised features are maintained is therefore crucial. By analysing the function of the clathrin adaptor AP-1 in the C. elegans intestine, we found that AP-1 is required for epithelial polarity maintenance. Depletion of AP-1 subunits does not affect epithelial polarity establishment or the formation of the intestinal lumen. However, the loss of AP-1 affects the polarised distribution of both apical and basolateral transmembrane proteins. Moreover, it triggers de novo formation of ectopic apical lumens between intestinal cells along the lateral membranes later during embryogenesis. We also found that AP-1 is specifically required for the apical localisation of the small GTPase CDC-42 and the polarity determinant PAR-6. Our results demonstrate that AP-1 controls an apical trafficking pathway required for the maintenance of epithelial polarity in vivo in a tubular epithelium

Plasmodesmata Ultrastructure Determination Using Electron Tomography.

Plant plasmodesmata (PD) are complex intercellular channels consisting of a thin endoplasmic reticulum (ER) tubule enveloped by the plasma membrane (PM). PD were first observed by electron microscopy about 50 years ago and, since, numerous studies in transmission and scanning electron microscopy have provided important information regarding their overall organization, revealing at the same time their diversity in terms of structure and morphology. However, and despite the fact that PD cell-cell communication is of critical importance for plant growth, development, cellular patterning, and response to biotic and abiotic stresses, linking their structural organization to their functional state has been proven difficult. This is in part due to their small size (20-50 nm in diameter) and the difficulty to resolve these structures in three dimensions at nanometer resolution to provide details of their internal organization.In this protocol, we provide in detail a complete process to produce high-resolution transmission electron tomograms of PD. We describe the preparation of the plant sample using high-pressure cryofixation and cryo-substitution. We also describe how to prepare filmed grids and how to cut and collect the sections using an ultramicrotome. We explain how to acquire a tilt series and how to reconstruct a tomogram from it using the IMOD software. We also give a few guidelines on segmentation of the reconstructed tomogram

Arabidopsis LDIP protein locates at a confined area within the lipid droplet surface and favors lipid droplet formation

International audienceLipid droplets (LDs) are cell organelles specialized in neutral lipid storage. Extendedly studied in seeds, LDs also accumulate in leaves during senescence or in response to abiotic stresses. However the mechanisms underlying their biogenesis remain relatively unknown. Here, we deciphered the distinct roles of two proteins during LD biogenesis: LD-associated protein 1 (AtLDAP1) and LDAP-interacting protein (AtLDIP). We demonstrated that AtLDIP overexpression favors the neo-formation of small LDs under growing conditions where LD accumulation is usually not observed. In addition, atldip knockout mutant displayed fewer but larger LDs, confirming a role of AtLDIP in LD biogenesis. Interestingly, a synergistic effect of the overexpression of both AtLDIP and AtLDAP1 was observed, resulting in an increase of LD cluster occurrence and LD abundance within the clusters and the cells. AtLDIP over-expression has no significant impact on triacylglycerol and steryl ester accumulation but AtLDIP inactivation is associated with an increase of neutral lipid content, that is probably a consequence of the enlarged but less abundant LDs present in this line. Our localization study demonstrated that AtLDIP is localized at specific dotted sites within the LD in contrast to AtLDAP1 that covers the whole LD. In addition, AtLDIP sometimes localized away from the LD marker, but always associated with the ER network, suggesting a location at LD nascent sites within the ER. Taken together, our results suggested that AtLDIP promotes the formation of new LDs from ER localized TAG lenses

A correlative light electron microscopy approach reveals plasmodesmata ultrastructure at the graft interface

Abstract Despite recent progress in our understanding of graft union formation, we still know little about the cellular events underlying the grafting process. This is partially due to the difficulty of reliably targeting the graft interface in electron microscopy to study its ultrastructure and three-dimensional architecture. To overcome this technological bottleneck, we developed a correlative light electron microscopy (CLEM) approach to study the graft interface with high ultrastructural resolution. Grafting hypocotyls of Arabidopsis thaliana lines expressing yellow FP or monomeric red FP in the endoplasmic reticulum (ER) allowed efficient targeting of the grafting interface for examination under light and electron microscopy. To explore the potential of our method to study sub-cellular events at the graft interface, we focused on the formation of secondary plasmodesmata (PD) between the grafted partners. We showed that four classes of PD were formed at the interface and that PD introgression into the cell wall was initiated equally by both partners. Moreover, the success of PD formation appeared not systematic with a third of PD not spanning the cell wall entirely. Characterizing the ultrastructural characteristics of these incomplete PD gives us insights into the process of secondary PD biogenesis. We found that the establishment of successful symplastic connections between the scion and rootstock occurred predominantly in the presence of thin cell walls and ER–plasma membrane tethering. The resolution reached in this work shows that our CLEM method advances the study of biological processes requiring the combination of light and electron microscopy