Damien De Bellis

Cryo-focused ion beam scanning electron microscopy (Cryo-FIBSEM) has become essential for preparing cryo-lamellae. Here, we present a series of improvements that speed up and enhance the efficiency of the Serial Lift-Out and SOLIST (Serialized On-grid Lift-In Sectioning for Tomography) procedures. We extend the cryo-FIBSEM session to 5 days and eliminate the need of copper or gold block intermediates and reduce curtaining effects. Finally, we report a routine to target lamellae with a precision of approximately 1 µm in X/Y/Z. We demonstrate the power of our improvements by targeting Casparian strips, suberin lamellae, as well as xylem vessels of Arabidopsis thaliana roots. This requires reaching a target of 5 micrometers in a 3 mm long and 80-120 µm thick root section. Despite ice formation in vacuoles and to some degree in the cytosol, plasma membranes and cell walls are remarkably well preserved, providing stunning insights into the native, hydrated nano-structure of plant cell walls, previously only observable with contrasting agents in a dehydrated state.

Cryo-focused ion beam scanning electron microscopy (cryo-FIBSEM) has become essential for preparing electron-transparent lamellae from cryo-plunged and high-pressure frozen specimens. However, targeting specific cellular features within large, complex organs remains challenging. Here we present a series of technical improvements significantly enhancing the efficiency and accessibility of the Serial Lift-Out and SOLIST (Serialized On-grid Lift-In Sectioning for Tomography) procedures that are revolutionizing the field. We were able to extend the cryo-FIBSEM session from 24 hours to 5 days without interruptions. In addition, we describe a modified silver-plated EasyLift ^TM needle that eliminates the need of the copper or gold block between the original tungsten needle and the sample. Moreover, we describe a strategy that significantly reduces curtaining effects. Finally, we report a precise routine to target a lamella with a precision of approximately 1 micrometer in X,Y and Z. Together, these modifications considerably reduce contamination risk and preparation time, making cryo-lift-out techniques more accessible for routine structural biology applications on any type of tissue. Here, we demonstrate the power of our technique by targeting several specific wall structures that are of crucial importance for root function in plants and that were previously inaccessible to cryo-electron tomography (cryo-ET). High-pressure freezing (HPF) of plant tissues presents unique challenges for cryo-electron microscopy sample preparation due to the overall sample size, the individual cells size, their rigid cell wall and finally, their large vacuoles, which contain large amounts of rather diluted water solutions compared to cytosol. The internal root structures targeted are the Casparian strip (CS), suberin lamellae (SL), as well as secondary wall of xylem vessels, requiring reaching a targeting precision of 5 micrometers in a 3 millimeters long and 80-120 micrometers thick root tip. Our technological improvements for the cryo-correlative light and electron microscopy (cryo-CLEM) workflow enabled successful, targeted cryo-ET in plant roots. We noticed that, despite ice formation in vacuoles and to some degree in the cytosol, the plasma membranes and cell walls are remarkably well preserved, providing stunning insights into the native, hydrated nano-structure of plant cell walls, previously only observable with contrasting agents and in a dehydrated state.

Summary: During the first days after germination, the root cap protects the seeding with its cuticle, which persists until the embryonic outer root cap cell layer is shed. The polyester constituting the delicate Arabidopsis root cap cuticle of the primary root has an atypical composition, containing unsubstituted very-long-chain fatty acids in addition to the oxygenated fatty acids typically found in cutin. The sn2-glycerol-3-phosphate acyltransferases GPAT4 and GPAT8, which possess an active phosphatase domain, synthesize monoacylglycerols and are crucial for incorporating oxygenated fatty acids into the root cap cutin. Here, we show that the incorporation of very-long-chain fatty acids into cutin requires the glycerol-3-phosphate acyltransferase GPAT2, which lacks an active phosphatase domain and produces lysophosphatidic acids. The GPAT2 knockout alters the properties of the root cap leading to increased mucilage deposition but lower barrier properties. Furthermore, we demonstrate that the long-chain CoA synthetase LACS2 is required solely for the incorporation of oxygenated fatty acids into cutin, similar to GPAT4 and GPAT8. Consequently, two largely non-redundant pathways contribute to root cap cuticle formation in the primary root: both the LACS2/GPAT4/GPAT8- and the GPAT2-dependent pathways contribute non-redundantly to its barrier and surface properties. LACS2 also plays an important role in the formation of the root cap cuticle in emerging lateral roots, whereas GPAT2 has a minor function in this process. These findings underscore the diverse mechanisms by which cuticle synthesis is fine-tuned across plant organs and developmental stages to achieve specialized barrier and surface properties.

Increasing soil salinity causes significant crop losses globally; therefore, understanding plant responses to salt (sodium) stress is of high importance. Plants avoid sodium toxicity through subcellular compartmentation by intricate processes involving a high level of elemental interdependence. Current technologies to visualize sodium, in particular, together with other elements, are either indirect or lack in resolution. Here we used the newly developed cryo nanoscale secondary ion mass spectrometry ion microprobe¹, which allows high-resolution elemental imaging of cryo-preserved samples and reveals the subcellular distributions of key macronutrients and micronutrients in root meristem cells of Arabidopsis and rice. We found an unexpected, concentration-dependent change in sodium distribution, switching from sodium accumulation in the cell walls at low external sodium concentrations to vacuolar accumulation at stressful concentrations. We conclude that, in root meristems, a key function of the NHX family sodium/proton antiporter SALT OVERLY SENSITIVE 1 (also known as Na⁺/H⁺ exchanger 7; SOS1/NHX7) is to sequester sodium into vacuoles, rather than extrusion of sodium into the extracellular space. This is corroborated by the use of new genomic, complementing fluorescently tagged SOS1 variants. We show that, in addition to the plasma membrane, SOS1 strongly accumulates at late endosome/prevacuoles as well as vacuoles, supporting a role of SOS1 in vacuolar sodium sequestration.

Multicellular organisms control environmental interactions through specialized barriers in specific cell types. A conserved barrier in plant roots is the endodermal Casparian strip (CS), a ring-like structure made of polymerized lignin that seals the endodermal apoplastic space. Most angiosperms have another root cell type, the exodermis, that is reported to form a barrier. Our understanding of exodermal developmental and molecular regulation and function is limited as this cell type is absent from Arabidopsis thaliana. We demonstrate that in tomato (Solanum lycopersicum), the exodermis does not form a CS. Instead, it forms a polar lignin cap (PLC) with equivalent barrier function to the endodermal CS but distinct genetic control. Repression of the exodermal PLC in inner cortical layers is conferred by the SlSCZ and SlEXO1 transcription factors, and these two factors genetically interact to control its polar deposition. Several target genes that act downstream of SlSCZ and SlEXO1 in the exodermis are identified. Although the exodermis and endodermis produce barriers that restrict mineral ion uptake, the exodermal PLC is unable to fully compensate for the lack of a CS. The presence of distinct lignin structures acting as apoplastic barriers has exciting implications for a root’s response to abiotic and biotic stimuli.

Cells harbour numerous receptor pathways to respond to diverse stimuli, yet often share common downstream signalling components. Mitogen-activated protein kinase (MPK) cascades are an example of such common hubs in eukaryotes. How such common hubs faithfully transduce distinct signals within the same cell-type is insufficiently understood, yet of fundamental importance for signal integration and processing in plants. We engineered a unique genetic background allowing direct comparisons of a developmental and an immunity pathway in one cell-type, the Arabidopsis root endodermis. We demonstrate that the two pathways maintain distinct functional and transcriptional outputs despite common MPK activity patterns. Nevertheless, activation of different MPK kinases and MPK classes led to distinct functional readouts, matching observed pathway-specific readouts. On the basis of our comprehensive analysis of core MPK signalling elements, we propose that combinatorial activation within the MPK cascade determines the differential regulation of an endodermal master transcription factor, MYB36, that drives pathway-specific gene activation.

Lipid polymers such as cutin and suberin strengthen the diffusion barrier properties of the cell wall in specific cell types and are essential for water relations, mineral nutrition, and stress protection in plants. Land plant–specific glycerol-3-phosphate acyltransferases (GPATs) of different clades are central players in cutin and suberin monomer biosynthesis. Here, we show that the GPAT4 / 6 / 8 clade in Arabidopsis thaliana , which is known to mediate cutin formation, is also required for developmentally regulated root suberization, in addition to the established roles of GPAT5/7 in suberization. The GPAT5 / 7 clade is mainly required for abscisic acid–regulated suberization. In addition, the GPAT5 / 7 clade is crucial for the formation of the typical lamellated suberin ultrastructure observed by transmission electron microscopy, as distinct amorphous globular polyester structures were deposited in the apoplast of the gpat5 gpat7 double mutant, in contrast to the thinner but still lamellated suberin deposition in the gpat4 gpat6 gpat8 triple mutant. Site-directed mutagenesis revealed that the intrinsic phosphatase activity of GPAT4, GPAT6, and GPAT8, which leads to monoacylglycerol biosynthesis, contributes to suberin formation. GPAT5/7 lack an active phosphatase domain and the amorphous globular polyester structure observed in the gpat5 gpat7 double mutant was partially reverted by treatment with a phosphatase inhibitor or the expression of phosphatase-dead variants of GPAT4 / 6 / 8. Thus, GPATs that lack an active phosphatase domain synthetize lysophosphatidic acids that might play a role in the formation of the lamellated structure of suberin. GPATs with active and nonactive phosphatase domains appear to have nonredundant functions and must cooperate to achieve the efficient biosynthesis of correctly structured suberin.

Summary: Lipid polymers such as cutin and suberin strengthen the diffusion barrier properties of the cell wall in specific cell types and are essential for water relations, mineral nutrition, and stress protection in plants. Land plant–specific glycerol-3-phosphate acyltransferases (GPATs) of different clades are central players in cutin and suberin monomer biosynthesis. Here, we show that the GPAT4 / 6 / 8 clade in Arabidopsis thaliana , which is known to mediate cutin formation, is also required for developmentally regulated root suberization, in addition to the established roles of GPAT5/7 in suberization. The GPAT5 / 7 clade is mainly required for abscisic acid–regulated suberization. In addition, the GPAT5 / 7 clade is crucial for the formation of the typical lamellated suberin ultrastructure observed by transmission electron microscopy, as distinct amorphous globular polyester structures were deposited in the apoplast of the gpat5 gpat7 double mutant, in contrast to the thinner but still lamellated suberin deposition in the gpat4 gpat6 gpat8 triple mutant. The intrinsic phosphatase activity of GPAT4, GPAT6, and GPAT8, which leads to monoacylglycerol biosynthesis, may be important for suberin biosynthesis. GPAT5/7 lack an active phosphatase domain. Notably, gpat5 gpat7 phenotypes were partially reverted by treatment with a phosphatase inhibitor or the expression of phosphatase-dead variants of GPAT4 / 6 / 8. Thus, GPATs that lack an active phosphatase domain, which are predicted to synthetize lysophosphatidic acids, might be crucial for the formation of the lamellated structure of suberin. GPATs with active and non-active phosphatase domains appear to have non-redundant functions and must cooperate to achieve the efficient biosynthesis of correctly structured suberin. Significance statement: The establishment of proper lamellated suberin in roots plays essential roles in regulating mineral nutrition and water relations in plants. The basis for the macromolecular arrangement determining the ultrastructure and properties of suberin lamellae is unknown. Here, we report that both the GPAT4 / 6 / 8 and GPAT5 / 7 clades of glycerol-3-phosphate acyltransferases (GPATs) contribute to suberin formation in Arabidopsis roots. In addition, we reveal that the GPAT5 / 7 clade is required for the formation of the lamellated suberin ultrastructure. Several lines of evidence suggest that the loss of phosphatase activity in GPATs might have played a crucial role in the formation of suberin lamellae during evolution.

Plant roots integrate environmental signals with development using exquisite spatiotemporal control. This is apparent in the deposition of suberin, an apoplastic diffusion barrier, which regulates flow of water, solutes and gases, and is environmentally plastic. Suberin is considered a hallmark of endodermal differentiation but is absent in the tomato endodermis. Instead, suberin is present in the exodermis, a cell type that is absent in the model organism Arabidopsis thaliana. Here we demonstrate that the suberin regulatory network has the same parts driving suberin production in the tomato exodermis and the Arabidopsis endodermis. Despite this co-option of network components, the network has undergone rewiring to drive distinct spatial expression and with distinct contributions of specific genes. Functional genetic analyses of the tomato MYB92 transcription factor and ASFT enzyme demonstrate the importance of exodermal suberin for a plant water-deficit response and that the exodermal barrier serves an equivalent function to that of the endodermis and can act in its place.

In plants, light direction is perceived by the phototropin photoreceptors, which trigger directional growth responses known as phototropism. The formation of a phototropin activation gradient across a photosensitive organ initiates this response. However, the optical tissue properties that functionally contribute to phototropism remain unclear. In this work, we show that intercellular air channels limit light transmittance through various organs in several species. Air channels enhance light scattering in Arabidopsis hypocotyls, thereby steepening the light gradient. This is required for an efficient phototropic response in Arabidopsis and Brassica. We identified an embryonically expressed ABC transporter required for the presence of air channels in seedlings and a structure surrounding them. Our work provides insights into intercellular air space development or maintenance and identifies a mechanism of directional light sensing in plants.