The endodermis: The building of a plant polarized epithelium
The endodermis is the innermost cortical cell layer of plant roots and surrounds their central vascular strand. It features Casparian strips, ring-like hydrophobic cell wall impregnations that surround each endodermal cells as a median belt and which are fused into a supracellular, net-like structure. This Casparian strip network represents the major extracellular (apoplastic) diffusion barrier in young roots. It serves to separate and protect the inner, extracellular space of the vascular cylinder from that of the cortex, which is continuous with the soil. Thereby, the endodermis is functionally equivalent to an animal polarized epithelium, such as the gut epithelium, for example. Very little was known in molecular terms about the building of this intricately structured cell layer that has evolved independently from animal epithelia. In a series of publication in recent years, we were able to describe the progression of endodermal differentiation and obtain markers that reveal the presence of a median, ring-like, lateral diffusion barrier in the plasma membrane, now termed the “Casparian strip domain” (CSD), as well as a strict polarity within the endodermis (see Alassimone et al., PNAS, 2010). We identified a previously uncharacterized family of proteins, now named “CAsparian Strip domain Proteins” (CASPs) as well as “CASP-Likes” (CASPLs) for the extended family of CASP-related proteins (see Roppolo et al., Plant Physiol., 2014). We could demonstrate that the CASPs are functionally equivalent to animal tight junction proteins in that they set up a median domain that acts as a lateral diffusion barrier as well as the determining the position of the Casparian strip cell wall impregnation itself (see Roppolo et al., Nature, 2011). We clarified a long-standing debate about the molecular nature of this cell wall impregnation, demonstrating that the Casparian strips are made of lignin and not suberin, as often presumed (see Naseer et al., PNAS, 2012). Based on this finding we then obtained mechanistic insights into how the CASPs provide a transmembrane protein scaffold that assembles what we propose is a “lignin polymerizing complex”, consisting of a specific NADPH oxidase, as well as peroxidases. We postulate that many other proteins – such as ESB1, identified by David Salt’s group (Hosmani et al., PNAS) – and other, as yet unidentified proteins are part of the lignin polymerising complex. Our model explains how precise subcellular polymerization of lignin in a centrally located belt might be achieved (Lee et al., Cell, 2012). In two consecutive forward genetic screens, we identified a number of genes necessary for Casparian strip formation (Alassimone, Fujita et al., Nat Plants, 2016 and Kalmbach et al., Nat Plants, 2017). A breakthrough in our understanding of endodermal differentiation came when we realised that four of the five SCHENGEN mutants, identified in our first genetic screen appear to constitute a novel signal transduction pathway which effectively sets up a “barrier surveillance” mechanism ensuring that the supracellular Casparian strip network will be effectively sealed (Doblas et al., Science, 2017; Alassimone, Fujita et al, Nat Plants, 2016, Pfister et al., eLife, 2014). This is achieved by an elegant separation of secreted ligand, produced in the stele, and a receptor signalling module confined to the cortex-facing plasma membrane domain of the endodermis. Such a setup ensures that signalling will only cease once any discontinuity in the Casparian strip has been sealed (Doblas et al., Science, 2017). In our second, LORD OF THE RINGS (LOTR) screen, in which we directly scored for mislocalisation of our CASP1-GFP marker, we identified numerous additional mutants, one of which lotr2 is an allele of EXO70A1, a specific exocyst subunit. Interestingly, this mutant does not affect secretion of CASP1-GFP, but leads to a complete mis-localisation of CASP1-GFP into a multitude of small microdomain at the plasma membrane, demonstrating that specific EXO70 subunits in plants are used as localised landmarks for specific secretion processes (Kalmbach et al., Nat Plants, 2017). Another mutant, lotr1, was characterised by Toru Fujiwara’s group (Li et al., Current Biology, 2017) and encodes a secreted protein of unknown function. We have written a number of reviews that summarise and further discuss diverse aspects of our work and of endodermal development in general (see our reviews).
The functional relevance of the endodermis
Our molecular and cell biological investigations of endodermal differentiation has now provided us with a number of specific mutants in endodermal barrier formation that we think are invaluable novel tools to finally understand the many supposed roles of the endodermis in root function, specifically its role in the selective uptake and retention of nutrients, but also its proposed function as a protective barrier to pathogens, for example. Using the sgn3 mutant, which show severely disrupted Casparian strips, we could demonstrate that this mutant has complex changes in its leaf ionomic profile. Among the many nutrients affected, a very robust reduction in potassium levels appears to be in large parts responsible for the overall growth reduction and low potassium hypersensitivity of the mutant (Pfister et al., eLife, 2014).
More recently, we have focused more attention on endodermal suberisation, a well described “secondary state” of endodermal differentiation in which endodermal cells surround themselves with a coat of hydrophobic suberin. Suberisation is bound to have large impact on the ability of endodermal cells to sense biotic and abiotic stresses and to take up nutrients. Indeed, we could show that a number of distinct nutrient stresses either enhance or decrease endodermal suberisation, through increases in abscissic acid (ABA) or ethylene hormone, respectively. These changes in suberisation are physiologically adaptive in that they can provide for increased uptake or retention of a given element (Barberon et al, Cell, 2017 ). Intriguingly, even within a fully suberized endodermis, some cells are kept in an unsuberised state. Although, described for a long time, the development and function of these so-called “passage cells” has remained obscure. Recently, we could demonstrate that passage cells arise from a previously undetected bisymmetry in the endodermis, which can be subdivided into smaller, less cytokinin- and ABA-sensitive, xylem-pole endodermis and longer, more cytokinin- and ABA-sensitive phloem-pole endodermis. This difference arises in part from movement of a stele-expressed cytokinin repressor protein into the endodermis (Andersen et al., Nature, 2018 ). Moreover, we could demonstrate that phosphate transport genes are expressed specifically in passage cells and that the presence of passage cells can even influence expression of transport genes in surrounding cortex and epidermal cells. As for overall suberisation, nutrient stresses, such as low phosphate, influence the number of passage cells that are formed in the endodermis. For many aspects of nutrient physiology, we collaborate with experts on nutrient physiology, such as the laboratory of David Salt in Nottingham, as well as with the laboratory of Junpei Takano in Osaka, with which we had an HFSP Young Investigator grant.