A suberized exodermis is required for tomato drought tolerance.

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.

Physiological roles of Casparian strips and suberin in the transport of water and solutes.

The formation of Casparian strips (CS) and the deposition of suberin at the endodermis of plant roots are thought to limit the apoplastic transport of water and ions. We investigated the specific role of each of these apoplastic barriers in the control of hydro-mineral transport by roots and the consequences on shoot growth. A collection of Arabidopsis thaliana mutants defective in suberin deposition and/or CS development was characterized under standard conditions using a hydroponic system and the Phenopsis platform. Mutants altered in suberin deposition had enhanced root hydraulic conductivity, indicating a restrictive role for this compound in water transport. In contrast, defective CS directly increased solute leakage and indirectly reduced root hydraulic conductivity. Defective CS also led to a reduction in rosette growth, which was partly dependent on the hydro-mineral status of the plant. Ectopic suberin was shown to partially compensate for defective CS phenotypes. Altogether, our work shows that the functionality of the root apoplastic diffusion barriers greatly influences the plant physiology, and that their integrity is tightly surveyed.

Transcriptomic Analysis of Resistant and Susceptible Responses in a New Model Root-Knot Nematode Infection System Using Solanum torvum and Meloidogyne arenaria.

Root-knot nematodes (RKNs) are among the most devastating pests in agriculture. Solanum torvum Sw. (Turkey berry) has been used as a rootstock for eggplant (aubergine) cultivation because of its resistance to RKNs, including Meloidogyne incognita and M. arenaria. We previously found that a pathotype of M. arenaria, A2-J, is able to infect and propagate in S. torvum. In vitro infection assays showed that S. torvum induced the accumulation of brown pigments during avirulent pathotype A2-O infection, but not during virulent A2-J infection. This experimental system is advantageous because resistant and susceptible responses can be distinguished within a few days, and because a single plant genome can yield information about both resistant and susceptible responses. Comparative RNA-sequencing analysis of S. torvum inoculated with A2-J and A2-O at early stages of infection was used to parse the specific resistance and susceptible responses. Infection with A2-J did not induce statistically significant changes in gene expression within one day post-inoculation (DPI), but afterward, A2-J specifically induced the expression of chalcone synthase, spermidine synthase, and genes related to cell wall modification and transmembrane transport. Infection with A2-O rapidly induced the expression of genes encoding class III peroxidases, sesquiterpene synthases, and fatty acid desaturases at 1 DPI, followed by genes involved in defense, hormone signaling, and the biosynthesis of lignin at 3 DPI. Both isolates induced the expression of suberin biosynthetic genes, which may be triggered by wounding during nematode infection. Histochemical analysis revealed that A2-O, but not A2-J, induced lignin accumulation at the root tip, suggesting that physical reinforcement of cell walls with lignin is an important defense response against nematodes. The S. torvum-RKN system can provide a molecular basis for understanding plant-nematode interactions.

Tissue-Autonomous Phenylpropanoid Production Is Essential for Establishment of Root Barriers.

Plants deposit hydrophobic polymers, such as lignin or suberin, in their root cell walls to protect inner tissues and facilitate selective uptake of solutes. Insights into how individual root tissues contribute to polymer formation are important for elucidation of ultrastructure, function, and development of these protective barriers. Although the pathways responsible for production of the barrier constituents are established, our models lack spatiotemporal resolution-especially in roots-thus, the source of monomeric barrier components is not clear. This is mainly due to our restricted ability to manipulate synthesis of the broadly important phenylpropanoid pathway, as mutants in this pathway display lethal or pleiotropic phenotypes. Here, we overcome this challenge by exploiting highly controlled in vivo repression systems. We provide strong evidence that autonomous production of phenylpropanoids is essential for establishment of the endodermal Casparian strip as well as adherence of the suberin matrix to the cell wall of endodermis and cork. Our work highlights that, in roots, the phenylpropanoid pathway is under tight spatiotemporal control and serves distinct roles in barrier formation across tissues and developmental zones. This becomes evident in the late endodermis, where repression of phenylpropanoid production leads to active removal of suberin in pre-suberized cells, indicating that endodermal suberin depositions might embody a steady state between continuous synthesis and degradation.

Spatio-temporal control of phenylpropanoid biosynthesis by inducible complementation of a cinnamate 4-hydroxylase mutant.

Cinnamate 4-hydroxylase (C4H) is a cytochrome P450-dependent monooxygenase that catalyzes the second step of the general phenylpropanoid pathway. Arabidopsis reduced epidermal fluorescence 3 (ref3) mutants, which carry hypomorphic mutations in C4H, exhibit global alterations in phenylpropanoid biosynthesis and have developmental abnormalities including dwarfing. Here we report the characterization of a conditional Arabidopsis C4H line (ref3-2pOpC4H), in which wild-type C4H is expressed in the ref3-2 background. Expression of C4H in plants with well-developed primary inflorescence stems resulted in restoration of fertility and the production of substantial amounts of lignin, revealing that the developmental window for lignification is remarkably plastic. Following induction of C4H expression in ref3-2pOpC4H, we observed rapid and significant reductions in the levels of numerous metabolites, including several benzoyl and cinnamoyl esters and amino acid conjugates. These atypical conjugates were quickly replaced with their sinapoylated equivalents, suggesting that phenolic esters are subjected to substantial amounts of turnover in wild-type plants. Furthermore, using localized application of dexamethasone to ref3-2pOpC4H, we show that phenylpropanoids are not transported appreciably from their site of synthesis. Finally, we identified a defective Casparian strip diffusion barrier in the ref3-2 mutant root endodermis, which is restored by induction of C4H expression.

Coordination between microbiota and root endodermis supports plant mineral nutrient homeostasis.

Plant roots and animal guts have evolved specialized cell layers to control mineral nutrient homeostasis. These layers must tolerate the resident microbiota while keeping homeostatic integrity. Whether and how the root diffusion barriers in the endodermis, which are critical for the mineral nutrient balance of plants, coordinate with the microbiota is unknown. We demonstrate that genes controlling endodermal function in the model plant Arabidopsis thaliana contribute to the plant microbiome assembly. We characterized a regulatory mechanism of endodermal differentiation driven by the microbiota with profound effects on nutrient homeostasis. Furthermore, we demonstrate that this mechanism is linked to the microbiota's capacity to repress responses to the phytohormone abscisic acid in the root. Our findings establish the endodermis as a regulatory hub coordinating microbiota assembly and homeostatic mechanisms.

Root endodermal barrier system contributes to defence against plant-parasitic cyst and root-knot nematodes.

Plant-parasitic nematodes (PPNs) cause tremendous yield losses worldwide in almost all economically important crops. The agriculturally most important PPNs belong to a small group of root-infecting sedentary endoparasites that includes cyst and root-knot nematodes. Both cyst and root-knot nematodes induce specialized long-term feeding structures in root vasculature from which they obtain their nutrients. A specialized cell layer in roots called the endodermis, which has cell walls reinforced with suberin deposits and a lignin-based Casparian strip (CS), protects the vascular cylinder against abiotic and biotic threats. To date, the role of the endodermis, and especially of suberin and the CS, during plant-nematode interactions was largely unknown. Here, we analyzed the role of suberin and CS during interaction between Arabidopsis plants and two sedentary root-parasitic nematode species, the cyst nematode Heterodera schachtii and the root-knot nematode Meloidogyne incognita. We found that nematode infection damages the endodermis leading to the activation of suberin biosynthesis genes at nematode infection sites. Although feeding sites induced by both cyst and root-knot nematodes are surrounded by endodermis during early stages of infection, the endodermis is degraded during later stages of feeding site development, indicating periderm formation or ectopic suberization of adjacent tissue. Chemical suberin analysis showed a characteristic suberin composition resembling peridermal suberin in nematode-infected tissue. Notably, infection assays using Arabidopsis lines with CS defects and impaired compensatory suberization, revealed that the CS and suberization impact nematode infectivity and feeding site size. Taken together, our work establishes the role of the endodermal barrier system in defence against a soil-borne pathogen.

Surveillance of cell wall diffusion barrier integrity modulates water and solute transport in plants.

The endodermis is a key cell layer in plant roots that contributes to the controlled uptake of water and mineral nutrients into plants. In order to provide such functionality the endodermal cell wall has specific chemical modifications consisting of lignin bands (Casparian strips) that encircle each cell, and deposition of a waxy-like substance (suberin) between the wall and the plasma membrane. These two extracellular deposits provide control of diffusion enabling the endodermis to direct the movement of water and solutes into and out of the vascular system in roots. Loss of integrity of the Casparian strip-based apoplastic barrier is sensed by the leakage of a small peptide from the stele into the cortex. Here, we report that such sensing of barrier integrity leads to the rebalancing of water and mineral nutrient uptake, compensating for breakage of Casparian strips. This rebalancing involves both a reduction in root hydraulic conductivity driven by deactivation of aquaporins, and downstream limitation of ion leakage through deposition of suberin. These responses in the root are also coupled to a reduction in water demand in the shoot mediated by ABA-dependent stomatal closure.

The Root Cap Cuticle: A Cell Wall Structure for Seedling Establishment and Lateral Root Formation.

The root cap surrounding the tip of plant roots is thought to protect the delicate stem cells in the root meristem. We discovered that the first layer of root cap cells is covered by an electron-opaque cell wall modification resembling a plant cuticle. Cuticles are polyester-based protective structures considered exclusive to aerial plant organs. Mutations in cutin biosynthesis genes affect the composition and ultrastructure of this cuticular structure, confirming its cutin-like characteristics. Strikingly, targeted degradation of the root cap cuticle causes a hypersensitivity to abiotic stresses during seedling establishment. Furthermore, lateral root primordia also display a cuticle that, when defective, causes delayed outgrowth and organ deformations, suggesting that it facilitates lateral root emergence. Our results show that the previously unrecognized root cap cuticle protects the root meristem during the critical phase of seedling establishment and promotes the efficient formation of lateral roots.

Cell wall composition and penetration resistance against the fungal pathogen Colletotrichum higginsianum are affected by impaired starch turnover in Arabidopsis mutants.

Penetration resistance represents the first level of plant defense against phytopathogenic fungi. Here, we report that the starch-deficient Arabidopsis thaliana phosphoglucomutase (pgm) mutant has impaired penetration resistance against the hemibiotrophic fungus Colletotrichum higginsianum. We could not determine any changes in leaf cutin and epicuticular wax composition or indolic glucosinolate levels, but detected complex alterations in the cell wall monosaccharide composition of pgm. Notably, other mutants deficient in starch biosynthesis (adg1) or mobilization (sex1) had similarly affected cell wall composition and penetration resistance. Glycome profiling analysis showed that both overall cell wall polysaccharide extractability and relative extractability of specific pectin and xylan epitopes were affected in pgm, suggesting extensive structural changes in pgm cell walls. Screening of mutants with alterations in content or modification of specific cell wall monosaccharides indicated an important function of pectic polymers for penetration resistance and hyphal growth of C. higginsianum during the biotrophic interaction phase. While mutants with affected pectic rhamnogalacturonan-I (mur8) were hypersusceptible, penetration frequency and morphology of fungal hyphae were impaired on pmr5 pmr6 mutants with increased pectin levels. Our results reveal a strong impact of starch metabolism on cell wall composition and suggest a link between carbohydrate availability, cell wall pectin and penetration resistance.

Environmental and physiological effects on grouping of drought-tolerant and susceptible rice varieties related to rice (Oryza sativa) root hydraulics under drought.

Background and Aims Root hydraulic limitations (i.e. intra-plant restrictions to water movement) may be related to crop performance under drought, and groupings in the hydraulic function of drought-tolerant and drought-susceptible rice (Oryza sativa) varieties have been previously reported. This study aimed to better understand the environmental and physiological relationships with rice root hydraulics under drought. Methods Xylem sap bleeding rates in the field (gsap g-1shoot) were measured on seasonal and diurnal time frames, during which time environmental conditions were monitored and physiological measurements were conducted. Complementary experiments on the effects of vapour pressure deficit (VPD) on root hydraulic conductivity and on transpiration rates of de-rooted tillers were conducted in growth chambers. Key Results The diurnal effects on bleeding rate were more closely related to irradiance than VPD, and VPD effects on root hydraulic conductivity measured on 21-day-old plants were due to effects on plant growth including root surface area, maximum root depth and root:shoot ratio. Leaf osmotic potential was related to the grouping of drought-tolerant and drought-susceptible varieties in rice root hydraulics, and these groupings were independent of differences in phenology. Low single-tiller bleeding rates were observed under high evapo-transpirational demand, higher bleeding rates were observed at more negative leaf osmotic potentials in drought-susceptible varieties, and drought-tolerant and susceptible varieties differed in the VPD-induced increase in transpiration rates of de-rooted tillers. Low root suberin amounts in some of the drought-susceptible varieties may have resulted in higher ion transport, as evidenced by higher sap K+ concentration and higher bleeding rates in those varieties. Conclusions These results provide evidence of the environmental effects on shoots that can influence root hydraulics. The consistent groupings of drought-tolerant and susceptible varieties suggest that traits affecting plant osmotic status may regulate root hydraulic response to drought in rice.

Wax and cutin mutants of Arabidopsis: Quantitative characterization of the cuticular transport barrier in relation to chemical composition.

Using (14)C-labeled epoxiconazole as a tracer, cuticular permeability of Arabidopsis thaliana leaves was quantitatively measured in order to compare different wax and cutin mutants (wax2, cut1, cer5, att1, bdg, shn3 and shn1) to the corresponding wild types (Col-0 and Ws). Mutants were characterized by decreases or increases in wax and/or cutin amounts. Permeances [ms(-1)] of Arabidopsis cuticles either increased in the mutants compared to wild type or were not affected. Thus, genetic changes in wax and cutin biosynthesis in some of the investigated Arabidopsis mutants obviously impaired the coordinated cutin and wax deposition at the outer leaf epidermal cell wall. As a consequence, barrier properties of cuticles were significantly decreased. However, increasing cutin and wax amounts by genetic modifications, did not automatically lead to improved cuticular barrier properties. As an alternative approach to the radioactive transport assay, changes in chlorophyll fluorescence were monitored after foliar application of metribuzine, an herbicide inhibiting electron transport in chloroplasts. Since both, half-times of photosynthesis inhibition as well as times of complete inhibition, in fact correlated with (14)C-epoxiconazole permeances, different rates of decline of photosynthetic yield between mutants and wild type must be a function of foliar uptake of the herbicide across the cuticle. Thus, monitoring changes in chlorophyll fluorescence, instead of conducting radioactive transport assays, represents an easy-to-handle and fast alternative evaluating cuticular barrier properties of different genotypes. This article is part of a Special Issue entitled: Plant Lipid Biology edited by Kent D. Chapman and Ivo Feussner.

Ester Cross-Link Profiling of the Cutin Polymer of Wild-Type and Cutin Synthase Tomato Mutants Highlights Different Mechanisms of Polymerization.

Cuticle function is closely related to the structure of the cutin polymer. However, the structure and formation of this hydrophobic polyester of glycerol and hydroxy/epoxy fatty acids has not been fully resolved. An apoplastic GDSL-lipase known as CUTIN SYNTHASE1 (CUS1) is required for cutin deposition in tomato (Solanum lycopersicum) fruit exocarp. In vitro, CUS1 catalyzes the self-transesterification of 2-monoacylglycerol of 9(10),16-dihydroxyhexadecanoic acid, the major tomato cutin monomer. This reaction releases glycerol and leads to the formation of oligomers with the secondary hydroxyl group remaining nonesterified. To check this mechanism in planta, a benzyl etherification of nonesterified hydroxyl groups of glycerol and hydroxy fatty acids was performed within cutin. Remarkably, in addition to a significant decrease in cutin deposition, mid-chain hydroxyl esterification of the dihydroxyhexadecanoic acid was affected in tomato RNA interference and ethyl methanesulfonate-cus1 mutants. Furthermore, in these mutants, the esterification of both sn-1,3 and sn-2 positions of glycerol was impacted, and their cutin contained a higher molar glycerol-to-dihydroxyhexadecanoic acid ratio. Therefore, in planta, CUS1 can catalyze the esterification of both primary and secondary alcohol groups of cutin monomers, and another enzymatic or nonenzymatic mechanism of polymerization may coexist with CUS1-catalyzed polymerization. This mechanism is poorly efficient with secondary alcohol groups and produces polyesters with lower molecular size. Confocal Raman imaging of benzyl etherified cutins showed that the polymerization is heterogenous at the fruit surface. Finally, by comparing tomato mutants either affected or not in cutin polymerization, we concluded that the level of cutin cross-linking had no significant impact on water permeance.

Partial depolymerization of genetically modified potato tuber periderm reveals intermolecular linkages in suberin polyester.

Suberin is a biopolyester found in specialized plant tissues, both internal and external, with key frontier physiological functions. The information gathered so far from its monomer and oligomer composition, and in situ studies made by solid state techniques, haven't solved the enigma of how the suberin polyester is assembled as a macromolecule. To investigate how monomers are linked in suberin, we analyzed oligomer fragments solubilized by the partial depolymerization of suberin from potato (Solanum tuberosum) tuber periderms. The structure of the suberin oligomers, namely which monomers they included, and the type and frequency of the inter-monomer ester linkages, was assessed by ESI-MS/MS and high resolution NMR analysis. The analyzed potato periderms included the one from wild type (cv. Desirée) and from plants where suberin-biosynthesis genes were downregulated in chain elongation (StKCS6), ω-hydroxylation (CYP86A33) and feruloylation (FHT). Two building blocks were identified as possible key structures in the macromolecular development of the potato periderm suberin: glycerol - α,ω-diacid - glycerol, as the core of a continuous suberin aliphatic polyester; and glycerol - ω-hydroxyacid - ferulic acid, anchoring this polyaliphatic matrix at its periphery to the vicinal polyaromatics, through linking to ferulic acid. The silencing of the StKCS6 gene led to non-significant alterations in suberin structure, showing the relatively minor role of the very-long chain (>C28) fatty acids in potato suberin composition. The silencing of CYP86A33 gene impaired significantly suberin production and disrupted the biosynthesis of acylglycerol structures, proving the relevance of the latter and thus of the glycerol - α,ω-diacid - glycerol unit for the typical suberin lamellar organization. The silencing of the FHT gene led to a lower frequency of ferulate linkages in suberin polyester but to more polyphenolic guaiacyl units as seen by FTIR analyses in the intact polymer.

Evolutionary conserved function of barley and Arabidopsis 3-KETOACYL-CoA SYNTHASES in providing wax signals for germination of powdery mildew fungi.

For plant pathogenic fungi, such as powdery mildews, that survive only on a limited number of host plant species, it is a matter of vital importance that their spores sense that they landed on the right spot to initiate germination as quickly as possible. We investigated a barley (Hordeum vulgare) mutant with reduced epicuticular leaf waxes on which spores of adapted and nonadapted powdery mildew fungi showed reduced germination. The barley gene responsible for the mutant wax phenotype was cloned in a forward genetic screen and identified to encode a 3-KETOACYL-CoA SYNTHASE (HvKCS6), a protein participating in fatty acid elongation and required for synthesis of epicuticular waxes. Gas chromatography-mass spectrometry analysis revealed that the mutant has significantly fewer aliphatic wax constituents with a chain length above C-24. Complementation of the mutant restored wild-type wax and overcame germination penalty, indicating that wax constituents less present on the mutant are a crucial clue for spore germination. Investigation of Arabidopsis (Arabidopsis thaliana) transgenic plants with sense silencing of Arabidopsis REQUIRED FOR CUTICULAR WAX PRODUCTION1, the HvKCS6 ortholog, revealed the same germination phenotype against adapted and nonadapted powdery mildew fungi. Our findings hint to an evolutionary conserved mechanism for sensing of plant surfaces among distantly related powdery mildews that is based on KCS6-derived wax components. Perception of such a signal must have been evolved before the monocot-dicot split took place approximately 150 million years ago.

RCN1/OsABCG5, an ATP-binding cassette (ABC) transporter, is required for hypodermal suberization of roots in rice (Oryza sativa).

Suberin is a complex polymer composed of aliphatic and phenolic compounds. It is a constituent of apoplastic plant interfaces. In many plant species, including rice (Oryza sativa), the hypodermis in the outer part of roots forms a suberized cell wall (the Casparian strip and/or suberin lamellae), which inhibits the flow of water and ions and protects against pathogens. To date, there is no genetic evidence that suberin forms an apoplastic transport barrier in the hypodermis. We discovered that a rice reduced culm number1 (rcn1) mutant could not develop roots longer than 100 mm in waterlogged soil. The mutated gene encoded an ATP-binding cassette (ABC) transporter named RCN1/OsABCG5. RCN1/OsABCG5 gene expression in the wild type was increased in most hypodermal and some endodermal roots cells under stagnant deoxygenated conditions. A GFP-RCN1/OsABCG5 fusion protein localized at the plasma membrane of the wild type. Under stagnant deoxygenated conditions, well suberized hypodermis developed in wild types but not in rcn1 mutants. Under stagnant deoxygenated conditions, apoplastic tracers (periodic acid and berberine) were blocked at the hypodermis in the wild type but not in rcn1, indicating that the apoplastic barrier in the mutant was impaired. The amount of the major aliphatic suberin monomers originating from C(28) and C(30) fatty acids or ω-OH fatty acids was much lower in rcn1 than in the wild type. These findings suggest that RCN1/OsABCG5 has a role in the suberization of the hypodermis of rice roots, which contributes to formation of the apoplastic barrier.

Acyl-lipid metabolism.

Acyl lipids in Arabidopsis and all other plants have a myriad of diverse functions. These include providing the core diffusion barrier of the membranes that separates cells and subcellular organelles. This function alone involves more than 10 membrane lipid classes, including the phospholipids, galactolipids, and sphingolipids, and within each class the variations in acyl chain composition expand the number of structures to several hundred possible molecular species. Acyl lipids in the form of triacylglycerol account for 35% of the weight of Arabidopsis seeds and represent their major form of carbon and energy storage. A layer of cutin and cuticular waxes that restricts the loss of water and provides protection from invasions by pathogens and other stresses covers the entire aerial surface of Arabidopsis. Similar functions are provided by suberin and its associated waxes that are localized in roots, seed coats, and abscission zones and are produced in response to wounding. This chapter focuses on the metabolic pathways that are associated with the biosynthesis and degradation of the acyl lipids mentioned above. These pathways, enzymes, and genes are also presented in detail in an associated website (ARALIP: http://aralip.plantbiology.msu.edu/). Protocols and methods used for analysis of Arabidopsis lipids are provided. Finally, a detailed summary of the composition of Arabidopsis lipids is provided in three figures and 15 tables.

Apoplastic diffusion barriers in Arabidopsis.

During the development of Arabidopsis and other land plants, diffusion barriers are formed in the apoplast of specialized tissues within a variety of plant organs. While the cuticle of the epidermis is the primary diffusion barrier in the shoot, the Casparian strips and suberin lamellae of the endodermis and the periderm represent the diffusion barriers in the root. Different classes of molecules contribute to the formation of extracellular diffusion barriers in an organ- and tissue-specific manner. Cutin and wax are the major components of the cuticle, lignin forms the early Casparian strip, and suberin is deposited in the stage II endodermis and the periderm. The current status of our understanding of the relationships between the chemical structure, ultrastructure and physiological functions of plant diffusion barriers is discussed. Specific aspects of the synthesis of diffusion barrier components and protocols that can be used for the assessment of barrier function and important barrier properties are also presented.

Casparian strip diffusion barrier in Arabidopsis is made of a lignin polymer without suberin.

Casparian strips are ring-like cell-wall modifications in the root endodermis of vascular plants. Their presence generates a paracellular barrier, analogous to animal tight junctions, that is thought to be crucial for selective nutrient uptake, exclusion of pathogens, and many other processes. Despite their importance, the chemical nature of Casparian strips has remained a matter of debate, confounding further molecular analysis. Suberin, lignin, lignin-like polymers, or both, have been claimed to make up Casparian strips. Here we show that, in Arabidopsis, suberin is produced much too late to take part in Casparian strip formation. In addition, we have generated plants devoid of any detectable suberin, which still establish functional Casparian strips. In contrast, manipulating lignin biosynthesis abrogates Casparian strip formation. Finally, monolignol feeding and lignin-specific chemical analysis indicates the presence of archetypal lignin in Casparian strips. Our findings establish the chemical nature of the primary root-diffusion barrier in Arabidopsis and enable a mechanistic dissection of the formation of Casparian strips, which are an independent way of generating tight junctions in eukaryotes.