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The biological function of the auxin phenylacetic acid (PAA) is not well characterized in plants. Although some aspects of its biology; transport, signaling and metabolism have recently been described. Previous work on this phytohormone has suggested that PAA behaves in an identical manner to IAA (indole-3-acetic acid) in promoting plant growth, yet plants require greater concentrations of PAA to elicit the same physiological responses. Here we show that normalized PAA treatment results in the differential expression of a unique list of genes, suggesting that plants can respond differently to the two auxins. This is further explored in endodermal barrier regulation where the two auxins invoke striking differences in the deposition patterns of suberin. We further show that auxin acts antagonistically on Casparian strip (CS) formation as it can circumvent the CS transcriptional machinery to repress CS related genes. Additionally, altered suberin biosynthesis reduces endogenous levels of PAA and CS deficiency represses the biosynthesis of IAA and the levels of both auxins. These findings implicate auxin as a regulator of endodermal barrier formation and highlight a novel role for PAA in root development and differentiation.

International audience; 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.

Microbes modify plant root permeability The root provides mineral nutrients and water to the plant. Diffusion barriers seal the root, preventing the loss of internal water and nutrients. Salas-González et al. found that microbes living on and in roots of the model plant Arabidopsis thaliana influence diffusion barrier formation, which affects the balance of mineral nutrients in the plant (see the Perspective by Busch and Chory). Plants with modified root diffusion barriers show altered bacterial community composition. Microbes tap into the plant's abscisic acid hormone signals to stabilize the root diffusion barrier against perturbations in environmental nutrient availability, thus enhancing plant stress tolerance. Science , this issue p. eabd0695 ; see also p. 125

Plants deposit polymeric barriers in their root cell walls to protect against external stress and facilitate selective nutrient uptake. The compounds that make up these barriers originate from the fatty acid- and phenylpropanoid biosynthetic pathways. Although the machinery responsible for production of the barrier constituents is well-char-acterized, our pathway models lack spatiotemporal resolution – especially in roots - and the source tissue is often not clear due to the apoplastic nature of barriers. Insights into how the individual root tissues or cells contribute to forming apoplastic barriers is important for elucidation of their ultrastructure, function and development. Manipulation of the associated biosynthesis is delicate, as mutants often display pleiotropic phenotypes due to the broad role of the underlying metabolites. Here, we address these issues by creating a genetic tool that allows in vivo repression of the phenylpropanoid pathway with both spatial and temporal control. We provide strong evidence that tissue-auton-omous production of phenylpropanoids is essential for establishment of the endodermal Casparian strip. Moreover, we find that in order to maintain deposition and attachment of a coherent suberin matrix to the cell wall, cells require continuous production of aromatic constituents. This process is especially crucial in the suberized endodermis where we find that repression of phenylpropanoid production leads to active removal of suberin.

Cinnamate 4-hydroxylase (C4H) is a cytochrome P450-dependent monooxygenase that catalyzes the second step of the general phenylpropanoid pathway. Arabidopsisreduced epidermal fluorescence 3(ref3) mutants, which carry hypomorphic mutations inC4H, 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-typeC4His expressed in theref3-2background. Expression ofC4Hin 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 ofC4Hexpression inref3-2pOpC4H, 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 toref3-2pOpC4H, we show that phenylpropanoids are not transported appreciably from their site of synthesis. Finally, we identified a defective Casparian strip diffusion barrier in theref3-2mutant root endodermis, which is restored by induction ofC4Hexpression.HighlightThe work presented this paper provides evidence of metabolite turnover, plasticity of the developmental window for lignification, and the impact of reduced and restored cinnamate-4-hydroxylase (C4H)expression on the Casparian strip.

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 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.

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.

International audience; 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.

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.

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 This work was supported by the Plant-KBBE SUIT Project (FCT grant PKBBE/AGR-GPL/0002/2009) and the grant from the Spanish Ministerio de Educación y Ciencia (AGL2006-07342).

Highlight Changes in Arabidopsis cell wall composition, predominantly in pectic polymers, influence penetration and establishment of Colletotrichum higginsianum hyphae in host leaves during initial biotrophy., 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.

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.

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.

Aliphatic alcohols naturally exist in many organisms as important cellular components; however, their roles in extracellular polymer biosynthesis are poorly defined. We report here the isolation and characterization of a rice (Oryza sativa) male-sterile mutant, defective pollen wall (dpw), which displays defective anther development and degenerated pollen grains with an irregular exine. Chemical analysis revealed that dpw anthers had a dramatic reduction in cutin monomers and an altered composition of cuticular wax, as well as soluble fatty acids and alcohols. Using map-based cloning, we identified the DPW gene, which is expressed in both tapetal cells and microspores during anther development. Biochemical analysis of the recombinant DPW enzyme shows that it is a novel fatty acid reductase that produces 1-hexadecanol and exhibits >270-fold higher specificity for palmiltoyl-acyl carrier protein than for C16:0 CoA substrates. DPW was predominantly targeted to plastids mediated by its N-terminal transit peptide. Moreover, we demonstrate that the monocot DPW from rice complements the dicot Arabidopsis thaliana male sterile2 (ms2) mutant and is the probable ortholog of MS2. These data suggest that DPWs participate in a conserved step in primary fatty alcohol synthesis for anther cuticle and pollen sporopollenin biosynthesis in monocots and dicots.

Suberin is an apoplastic biopolymer with tissue-specific deposition in the cell walls of the endo- and exodermis of roots, of periderms including wound periderm and other border tissues. Suberised cell walls contain both polyaliphatic and polyaromatic domains which are supposedly cross-linked. The predominant aliphatic components are ω-hydroxyacids, α,ω-diacids, fatty acids and primary alcohols, whereas hydroxycinnamic acids, especially ferulic acid, are the main components of the polyaromatic domain. Although the monomeric composition of suberin has been known for decades, its biosynthesis and deposition has mainly been a subject of speculation. Only recently, significant progress elucidating suberin biosynthesis has been achieved using molecular genetic approaches, especially in the model species Arabidopsis. In parallel, the long-standing hypothesis that suberin functions as an apoplastic barrier has been corroborated by sophisticated, quantitative physiological studies in the past decade. These studies demonstrated that suberised cell walls could act as barriers, minimising the movement of water and nutrients, restricting pathogen invasion and impeding toxic gas diffusion. In addition, suberised cell walls provide a barrier to radial oxygen loss from roots to the anaerobic root substrate in wetland plants. The recent onset of multidisciplinary approaches combining genetic, analytical and physiological studies has begun to deliver further insights into the physiological importance of suberin depositions in plants.

The effects of air pollutants such as aldehydes, ozone, nitrogen dioxide and benzene on fatty acid ω-hydroxylase activity in Vicia sativa microsomes have been investigated. Four days old etiolated V. sativa seedlings were exposed to different concentrations of selected pollutants for varying exposure times. Growing etiolated V. sativa seedlings in air containing the gaseous benzaldehyde (150 nM) led to an 8-fold enhancement of lauric acid ω-hydroxylase activity in microsomes of treated plants compared to controls grown in pure air (96±10 versus 12±2 pmol/min/mg protein, respectively). The induction increased with increasing gas phase concentrations (10–1300 nM) and the maximum of activity was measured after 48 h of exposure. Northern blot analysis revealed that this induction occurred via transcriptional activation of the gene coding for CYP94A1. The absence of CYP94A2 and CYP94A3 transcription activation together with the missing effect on epoxide hydrolases activities indicate the specificity of CYP94A1 induction by benzaldehyde. Exposure to nitrogen dioxide, ozone and formaldehyde also stimulated lauric acid ω-hydroxylases activity while exposure to benzene did not show any effect.

Plant cell walls are dramatically affected by suberin deposition, becoming an impermeable barrier to water and pathogens. Suberin is a complex layered heteropolymer that comprises both a poly(aliphatic) and a poly(aromatic) lignin-like domain. Current structural models for suberin attribute the crosslinking of aliphatic and aromatic domains within the typical lamellar ultrastructure of the polymer to esterified ferulate. BAHD feruloyl transferases involved in suberin biosynthesis have been recently characterized in Arabidopsis and potato (Solanum tuberosum). In defective mutants, suberin, even lacks most of the esterified ferulate, but maintains the typical lamellar ultrastructure. However, suberized tissues display increased water permeability, in spite of exhibiting a similar lipid load to wild type. Therefore, the role of ferulate in suberin needs to be reconsidered. Moreover, silencing the feruloyl transferase in potato turns the typical smooth skin of cv. Desirée into a rough scabbed skin distinctive of Russet varieties and impairs the normal skin maturation that confers resistance to skinning. Concomitantly to these changes, the skin of silenced potatoes shows an altered profile of soluble phenolics with the emergence of conjugated polyamines.

Apart from its significance in the protection against stress conditions, the cuticular cover is essential for proper development of the diverse surface structures formed on aerial plant organs. This layer mainly consists of a cutin matrix, embedded and overlaid with cuticular waxes. Following their biosynthesis in epidermal cells, cutin and waxes were suggested to be exported across the plasma membrane by ABCG-type transporters such as DSO/ABCG11 to the cell wall and further to extracellular matrix. Here, additional aspects of DSO/ABCG11 function were investigated, predominantly in reproductive organs, which were not revealed in the previous reports. This was facilitated by the generation of a transgenic DSO/ABCG11 silenced line (dso-4) that displayed relatively subtle morphological and chemical phenotypes. These included altered petal and silique morphology, fusion of seeds, and changes in levels of cutin monomers in flowers and siliques. The dso-4 phenotypes corresponded to the strong DSO/ABCG11 gene expression in the embryo epidermis as well as in the endosperm tissues of the developing seeds. Moreover, the DSO/ABCG11 protein displayed polar localization in the embryo protoderm. Transcriptome analysis of the dso-4 mutant leaves and stems showed that reduced DSO/ABCG11 activity suppressed the expression of a large number of cuticle-associated genes, implying that export of cuticular lipids from the plasma membrane is a rate-limiting step in cuticle metabolism. Surprisingly, root suberin composition of dso-4 was altered, as well as root expression of two suberin biosynthetic genes. Taken together, this study provides new insights into cutin and suberin metabolism and their role in reproductive organs and roots development.

Suberin and waxes embedded in the suberin polymer are key compounds in the control of transpiration in the tuber periderm of potato (Solanum tuberosum). Suberin is a cell-wall biopolymer with aliphatic and aromatic domains. The aliphatic suberin consists of a fatty acid polyester with esterified ferulic acid, which is thought to play an important role in cross-linking to the aromatic domain. In potato, ferulic acid esters are also the main components of periderm wax. How these ferulate esters contribute to the periderm water barrier remains unknown. Here we report on a potato gene encoding a fatty omega-hydroxyacid/fatty alcohol hydroxycinnamoyl transferase (FHT), and study its molecular and physiological relevance in the tuber periderm by means of a reverse genetic approach. In FHT RNAi periderm, the suberin and its associated wax contained much smaller amounts of ferulate esters, in agreement with the in vitro ability of the FHT enzyme to conjugate ferulic acid with omega-hydroxyacid and fatty alcohols. FHT down-regulation did not affect the typical suberin lamellar ultrastructure but had significant effects on the anatomy, sealing properties and maturation of the periderm. The tuber skin became thicker and russeted, water loss was greatly increased, and maturation was prevented. FHT deficiency also induced accumulation of the hydroxycinnamic acid amides feruloyl and caffeoyl putrescine in the periderm. We discuss these results in relation to the role attributed to ferulates in suberin molecular architecture and periderm impermeability.

Suberin is a hydrophobic polyester found in the cell walls of various plant-environment interfaces, including shoot and root peridermal tissue, and the root hypodermis and endodermis. Suberin deposits form apoplastic barriers that control water and nutrient transport, protect against pathogens and seal wounded tissue. Despite this physiological importance, and the detailed information on the suberin composition of many plants, there is a great gap in our knowledge of the molecular mechanism of suberin biosynthesis, caused in part by a lack of mutants in suberin formation. Here, we report the characterization of daisy, an Arabidopsis mutant that is defective in a fatty acid elongase condensing enzyme. The daisy mutant roots exhibit disturbed growth, and the suberin level is reduced in C(22) and C(24) very long chain fatty acid derivatives, whereas C(16), C(18) and C(20) derivatives accumulate, compared with wild-type suberin, indicating that DAISY functions as a docosanoic acid synthase. Consistent with a significantly increased level of suberin in the roots of NaCl-stressed plants, DAISY is transcriptionally activated by NaCl application, and also by polyethylene glycol-induced drought stress and wounding. Expression analysis using RT-PCR and promoter-GUS fusions demonstrated a distinct DAISY expression pattern in the root stele, senescing sepals, siliques abscission zones and the chalaza-micropyle region of seeds. Together, these results indicate that DAISY is involved in suberin biosynthesis and in the formation of protective layers in these tissues, and in the response to unfavourable environmental conditions.

Very long chain aliphatic compounds occur in the suberin polymer and associated wax. Up to now only few genes involved in suberin biosynthesis have been identified. This is a report on the isolation of a potato (Solanum tuberosum) 3-ketoacyl-CoA synthase (KCS) gene and the study of its molecular and physiological relevance by means of a reverse genetic approach. This gene, called StKCS6, was stably silenced by RNA interference (RNAi) in potato. Analysis of the chemical composition of silenced potato tuber periderms indicated that StKCS6 down-regulation has a significant and fairly specific effect on the chain length distribution of very long-chain fatty acids (VLCFAs) and derivatives, occurring in the suberin polymer and peridermal wax. All compounds with chain lengths of C(28) and higher were significantly reduced in silenced periderms, whereas compounds with chain lengths of C(26) and lower accumulated. Thus, StKCS6 is preferentially involved in the formation of suberin and wax lipidic monomers with chain lengths of C(28) and higher. As a result, peridermal transpiration of the silenced lines was about 1.5-times higher than that of the wild type. Our results convincingly show that StKCS6 is involved in both suberin and wax biosynthesis and that a reduction of the monomeric carbon chain lengths leads to increased rates of peridermal transpiration.

Suberin is a cell wall lipid polyester found in the cork cells of the periderm offering protection against dehydration and pathogens. Its biosynthesis and assembly, as well as its contribution to the sealing properties of the periderm, are still poorly understood. Here, we report on the isolation of the coding sequence CYP86A33 and the molecular and physiological function of this gene in potato (Solanum tuberosum) tuber periderm. CYP86A33 was down-regulated in potato plants by RNA interference-mediated silencing. Periderm from CYP86A33-silenced plants revealed a 60% decrease in its aliphatic suberin load and greatly reduced levels of C18:1 ω-hydroxyacid (approximately 70%) and α,ω-diacid (approximately 90%) monomers in comparison with wild type. Moreover, the glycerol esterified to suberin was reduced by 60% in the silenced plants. The typical regular ultrastructure of suberin, consisting of dark and light lamellae, disappeared and the thickness of the suberin layer was clearly reduced. In addition, the water permeability of the periderm isolated from CYP86A33-silenced lines was 3.5 times higher than that of the wild type. Thus, our data provide convincing evidence for the involvement of ω-functional fatty acids in establishing suberin structure and function.

Cuticular waxes, forming the plant/atmosphere interface of plants colonizing the terrestrial environment, are complex mixtures of very-long chain fatty acids (VLCFAs) and their derivatives. In VLCFAs biosynthesis, beta-ketoacyl CoA synthase (E.C.2.3.1.119, KCS) is the key enzyme. Using T-DNA insertional mutagenesis, we identified a cuticle-deficient rice mutant, which displayed a pleiotropic phenotype including reduced growth, leaf fusion, sparse wax crystals, enhanced sensitivity to drought and low fertility. Further analysis indicated that T-DNA was inserted in the 5'-UTR intron of the affected gene, Wax Crystal-Sparse Leaf1 (WSL1), and abnormal transcript caused the loss-of-function of WSL1 gene. Genetic complementation experiment confirmed the function of the candidate gene. WSL1 was predicted to encode a polypeptide containing a conserved FAE1_CUT1_RppA domain typical of the KCS family proteins. Qualitative and quantitative wax composition analyses by gas chromatography-mass spectrometry (GC-MS) demonstrated a marked reduction of total cuticular wax load on wsl1 leaf blades and sheaths, and VLCFA precursors of C20-C24 decreased in both. Moreover, ubiquitous expression of the WSL1 gene gave a hint that WSL1-catalyzed elongation of VLCFAs might participate in a wide range of rice growth and development processes beyond biosynthesis of cuticular waxes.

The lipophilic biopolyester suberin forms important boundaries to protect the plant from its surrounding environment or to separate different tissues within the plant. In roots, suberin can be found in the cell walls of the endodermis and the hypodermis or periderm. Apoplastic barriers composed of suberin accomplish the challenge to restrict water and nutrient loss and prevent the invasion of pathogens. Despite the physiological importance of suberin and the knowledge of the suberin composition of many plants, very little is known about its biosynthesis and the genes involved. Here, a detailed analysis of the Arabidopsis aliphatic suberin in roots at different developmental stages is presented. This study demonstrates some variability in suberin amount and composition along the root axis and indicates the importance of omega-hydroxylation for suberin biosynthesis. Using reverse genetics, the cytochrome P450 fatty acid omega-hydroxylase CYP86A1 (At5g58860) has been identified as a key enzyme for aliphatic root suberin biosynthesis in Arabidopsis. The corresponding horst mutants show a substantial reduction in omega-hydroxyacids with a chain lengthC(20), demonstrating that CYP86A1 functions as a hydroxylase of root suberized tissue. Detailed expression studies revealed a strong root specificity and a localized expression in the root endodermis. Transgenic expression of CYP86A1 fused to GFP distributed CYP86A1 to the endoplasmic reticulum, indicating that suberin monomer biosynthesis takes place in this sub-cellular compartment before intermediates are exported in the apoplast.

A fatty-acid-metabolizing enzyme from Arabidopsis thaliana, CYP94C1, belonging to the cytochrome P450 family was cloned and characterized. CYP94C1 was heterologously expressed in a Saccharomyces cerevisiae strain (WAT11) engineered for P450 expression. When recombinant yeast microsomes were incubated with lauric acid (C12:0) for 15 min, one major metabolite was formed. The product was purified and identified by GC/MS as 12-hydroxylauric acid. Longer incubation (40 min) led to the formation of an additional metabolite identified by GC/MS as dodecadioic acid. This diacid was also produced by incubation with 12-hydroxylauric acid. These compounds were not produced by incubating microsomes from yeast transformed with a void plasmid, demonstrating the involvement of CYP94C1. This new enzyme also metabolized fatty acids of varying aliphatic chain lengths (C12 to C18) and in-chain modifications, for example, degree of unsaturation or the presence of an epoxide as an additional polar functional group. Transcription of the gene encoding CYP94C1 is enhanced by stress, treatment with the hormone methyl jasmonate and wounding. Treatment with methyl jasmonate also induced lauric acid metabolism in microsomes prepared from Arabidopsis. The induction of hydroxylase activity was dose dependent and increased with exposure time, reaching 16x higher in microsomes from 24-h treated Arabidopsis compared with control plants. Analysis of the metabolites showed a mixture of 12-, 11- and 10-hydroxylauric acids, revealing for the first time the presence of fatty acid in-chain hydroxylase in Arabidopsis.

Suberized cell walls form physiologically important plant-environment interfaces because they act as barriers that limit water and nutrient transport and protect plants from invasion by pathogens. Plants respond to environmental stimuli by modifying the degree of suberization in root cell walls. Salt stress or drought-induced suberization leads to a decrease in radial water transport in roots. Although reinforced, suberized cell walls never act as absolutely impermeable barriers. Deeper insights into the structure and biosynthesis of suberin are required to elucidate what determines the barrier properties. Progress has been obtained from analytical methods that enabled the structural characterization of oligomeric building blocks in suberin, and from the opening of suberin research to molecular genetic approaches by the elucidation of the chemical composition and tissue distribution of suberin in the model species Arabidopsis.

The composition of wax and cutin from developing sweet cherry (Prunus avium) fruit was studied by GC-MS between 22 and 85 days after full bloom (DAFB). In this and our previous study, fruit mass and surface area increased in a sigmoidal pattern with time, but mass of the cuticular membrane (CM) per unit fruit surface area decreased. On a whole fruit basis, mass of CM increased up to 36 DAFB and remained constant thereafter. At maturity, triterpenes, alkanes and alcohols accounted for 75.6%, 19.1% and 1.2% of total wax, respectively. The most abundant constituents were the triterpenes ursolic (60.0%) and oleanolic acid (7.5%), the alkanes nonacosane (13.0%) and heptacosane (3.0%), and the secondary alcohol nonacosan-10-ol (1.1%). In developing fruit triterpenes per unit area decreased, but alkanes and alcohols remained essentially constant. The cutin fraction of mature fruit consisted of mostly C16 (69.5%) and, to a lower extent, C18 monomers (19.4%) comprising alkanoic, omega-hydroxyacids, alpha,omega-dicarboxylic and midchain hydroxylated acids. The most abundant constituents were 9(10),16-dihydroxy-hexadecanoic acid (53.6%) and 9,10,18-trihydroxy-octadecanoic acid (7.8%). Amounts of C16 and C18 monomers per unit area decreased in developing fruit, but remained approximately constant on a whole fruit basis. Within both classes of monomers, opposing changes occurred. Amounts of hexadecandioic, 16-hydroxy-hexadecanoic, 9(10)-hydroxy-hexadecane-1,16-dioic and 9,10-epoxy-octadecane-1,18-dioic acids increased, but 9,10,18-trihydroxy-octadecanoic and 9,10,18-trihydroxy-octadecenoic acids decreased. There were no qualitative and minor quantitative differences in wax and cutin composition between cultivars at maturity. Our data indicate that deposition of some constituents of wax and cutin ceased during early fruit development.

Cell walls are essential features of plant cells and modifications of this apoplastic space implement a number of structural and physiological functions. The Casparian strip (CS), named after its 1865 discoverer Robert Caspary (1), is a remarkable cell wall modification that all biology students become acquainted with in undergraduate plant anatomy courses. Typically, CSs occur in the endodermis of roots, the organ performing the uptake of water and nutrients. The CS can be envisioned as a belt-like structure, specifically localized in the middle of the anticlinal wall all around an endodermal cell (Fig. 1). CSs, chemically dominated by lignin (2, 3), bridge the cell wall of adjacent endodermal cells and thus seal the extracellular space. Consequently, apoplastic passage of ions is restricted and transporter and channel proteins in stele and soil-directed plasma membranes selectively permit the radial transport of ions from the soil to the stele (4). Conversely, CSs in the endodermis also prevent the apoplastic backflow of ions from the stele to the cortex. The resulting ion build-up in the stele and subsequent osmotic water movements generate the root pressure, enabling vertical water and nutrient transport when transpiration is limited. When roots develop further, suberin, a cell wall modification of aromatic and aliphatic materials, is deposited as lamella all around the endodermal cell (5). This additionally prevents transcellular paths across membranes and thus complicates the evaluation of the CS's impact on water relations and nutrient homeostasis. However, although a consistent root structure and physiologically essential, little is known about the molecular components that regulate CS localization and biogenesis. In PNAS, Kamiya et al. (6) present a great step forward in the understanding of how and what controls CS formation.

The developing leaf three of barley provides an excellent model system for the direct determination of relationships between amounts of waxes and cutin and cuticular permeance. Permeance of the cuticle was assessed via the time-course of uptake of either toluidine blue or (14)C-labelled benzoic acid ([(14)C] BA) along the length of the developing leaf. Toluidine blue uptake only occurred within the region 0-25 mm from the point of leaf insertion (POLI). Resistance--the inverse of permeance--to uptake of [(14)C] BA was determined for four leaf regions and was lowest in the region 10-20 mm above POLI. At 20-30 and 50-60 mm above POLI, it increased by factors of 6 and a further 32, respectively. Above the point of emergence of leaf three from the sheath of leaf two, which was 76-80 mm above POLI, resistance was as high as at 50-60 mm above POLI. GC-FID/MS analyses of wax and cutin showed that: (1) the initial seven fold increase in cuticular resistance coincided with increase in cutin coverage and appearance of waxes; (2) the second, larger and final increase in cuticle resistance was accompanied by an increase in wax coverage, whereas cutin coverage remained unchanged; (3) cutin deposition in barley leaf epidermis occurred in parallel with cell elongation, whereas deposition of significant amounts of wax commenced as cells ceased to elongate.

The outermost epidermal cell wall is specialized to withstand pathogens and natural stresses, and lipid-based cuticular polymers are the major barrier against incursions. The Arabidopsis thaliana mutant bodyguard (bdg), which exhibits defects characteristic of the loss of cuticle structure not attributable to a lack of typical cutin monomers, unexpectedly accumulates significantly more cell wall–bound lipids and epicuticular waxes than wild-type plants. Pleiotropic effects of the bdg mutation on growth, viability, and cell differentiation are also observed. BDG encodes a member of the α/β-hydrolase fold protein superfamily and is expressed exclusively in epidermal cells. Using Strep-tag epitope-tagged BDG for mutant complementation and immunolocalization, we show that BDG is a polarly localized protein that accumulates in the outermost cell wall in the epidermis. With regard to the appearance and structure of the cuticle, the phenotype conferred by bdg is reminiscent of that of transgenic Arabidopsis plants that express an extracellular fungal cutinase, suggesting that bdg may be incapable of completing the polymerization of carboxylic esters in the cuticular layer of the cell wall or the cuticle proper. We propose that BDG codes for an extracellular synthase responsible for the formation of cuticle. The alternative hypothesis proposes that BDG controls the proliferation/differentiation status of the epidermis via an unknown mechanism.

Due to their complex structure with several chiral centres important anticancer agents are still extracted from plants and not synthesized chemically on a commercial scale. Sustainable bioproduction of the compounds of interest may be achieved by plant in vitro cultures. Undifferentiated callus and suspension cultures, which can be cultivated in large bioreactors easily, very often fail to accumulate the compounds of interest, whereas shoot and root cultures as well hairy roots normally produce the same compounds as in the appropriate organs. The production of anticancer compounds, such as the alkaloids vinblastine, vincristine, paclitaxel (Taxol®), camptothecin, or the lignan podophyllotoxin, by plant in vitro cultures is reviewed. Taxanes can be produced in bioreactors using cell suspensions of various Taxus species with good yields; presently paclitaxel is produced on a commercial scale by Phyton Biotech (Germany). Camptothecin has low yields in suspension cultures of Camptotheca acuminata or Nothapodytes foetida (0.0003–0.01%), but a good production (0.1–0.3% dry wt) in root and hairy root cultures of Ophiorrhiza pumila, O. mungos and C. acuminata. Podophyllotoxin can be produced in cell suspension and root as well as hairy root cultures of Podophyllum and various Linum species up to 130 mg/l (Linum album cell suspensions); its derivative 6-methoxypodophyllotoxin is accumulated in hairy roots of L. persicum up to about 500 mg/l. The in vitro production of dimeric indole alkaloids in Catharanthus roseus has failed so far both in undifferentiated and differentiated in vitro cultures. In cases where in vitro cultures show good yields, they can be employed in biotechnology for the sustainable production of valuable products.

Castor bean (Ricinus communis L.) plants were hydroponically cultivated to achieve NO3 deficiency (N starvation), salt stress (addition of 100 mM NaCl), or normal conditions. Endodermal (ECW) and rhizodermal and hypodermal cell walls (RHCW) were isolated enzymatically from roots, and suberin monomers were released by transesterification after solvent extraction. Aromatic and aliphatic suberin monomers were identified and quantified by gas chromatography and mass spectrometry. Between 90 and 95% of the released suberin monomers were linear, long-chain, aliphatic compounds (alcohols, acids, diacids, ω-hydroxy acids and 2-hydroxy acids) with an average chain length of 19 C-atoms. The remainder was an aromatic suberin fraction mainly composed of coumaric and ferulic acid. Suberin amounts were significantly increased in ECW and RHCW in the presence of NaCl. In contrast, N starvation led to significantly reduced levels of suberization in ECW and RHCW. It is concluded that R. communis plants reinforce their apoplastic transport barriers in roots in adaptation to NaCl stress in order to minimize NaCl uptake. Under conditions of N starvation the opposite occurs and plants reduce the suberization of their apoplastic transport barriers to facilitate nutrient uptake form the soil.

Chemical analysis of 4-day-old corn (Zea mays L.) root cell walls revealed that the lipophilic biopolymer suberin forms an important constituent of rhizodermal and hypodermal cell walls. Identified aliphatic monomers had chain lengths ranging from C16 to C26 and they belonged to 5 substance classes (omega-hydroxycarboxylic acids, 1,omega-dicarboxylic acids, 2-hydroxycarboxylic acids, carboxylic acids and alcohols) by which suberin is characterized. Biochemical experiments proved the occurrence of elongase activities in corn roots. Highest enzymatic activities were found in corn root microsomes, and major products synthesized by root elongases were elongated fatty acids with chain lengths ranging from C20 to C24. Preferred substrates of root elongases were acyl-CoAs of the chain length C18 and C20, whereas monounsaturated acyl-CoAs (C16:1 and C18:1) and acyl-CoAs of lower (C12-C16) and higher chain lengths (C22-C24) were rarely elongated. Elongase activities significantly decreased over the length (40 cm) of 10-day-old corn roots going from the young tip to the older base of the root. Thus, results presented here show the presence and activity of elongases in roots of plants.

Summary 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 C28 and C30 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.

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.

Studying lignin-biosynthetic-pathway mutants and transgenics provides insights into plant responses to perturbations of the lignification system, and enhances our understanding of normal lignification. When enzymes late in the pathway are downregulated, significant changes in the composition and structure of lignin may result. NMR spectroscopy provides powerful diagnostic tools for elucidating structures in the difficult lignin polymer, hinting at the chemical and biochemical changes that have occurred. COMT (caffeic acid O-methyl transferase) downregulation in poplar results in the incorporation of 5-hydroxyconiferyl alcohol into lignins via typical radical coupling reactions, but post-coupling quinone methide internal trapping reactions produce novel benzodioxane units in the lignin. CAD (cinnamyl alcohol dehydrogenase) downregulation results in the incorporation of the hydroxycinnamyl aldehyde monolignol precursors intimately into the polymer. Sinapyl aldehyde cross-couples 8–O–4 with both guaiacyl and syringyl units in the growing polymer, whereas coniferyl aldehyde cross-couples 8–O–4 only with syringyl units, reflecting simple chemical cross-coupling propensities. The incorporation of hydroxycinnamyl aldehyde and 5-hydroxyconiferyl alcohol monomers indicates that these monolignol intermediates are secreted to the cell wall for lignification. The recognition that novel units can incorporate into lignins portends significantly expanded opportunities for engineering the composition and consequent properties of lignin for improved utilization of valuable plant resources.

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.

Suberin is a highly persistent cell wall polymer, predominantly composed of long-chain hydroxylated fatty acids. Apoplastic suberin depositions occur in internal and peripheral dermal tissues where they generate lipophilic barriers preventing uncontrolled flow of water, gases, and ions. In addition, suberization provides resistance to environmental stress conditions. Despite this physiological importance the knowledge about suberin formation has increased slowly for decades. Lately, the chemical characterization of suberin in Arabidopsis enabled the proposal of genes required for suberin biosynthesis such as β-ketoacyl-CoA synthases (KCS) for fatty acid elongation and cytochrome P450 oxygenases (CYP) for fatty acid hydroxylation. Advantaged by the Arabidopsis molecular genetic resources the in silico expression pattern of candidate genes, concerted with the tissue-specific distribution of suberin in Arabidopsis, led to the identification of suberin involved genes including KCS2, CYP86A1, and CYP86B1. The isolation of mutants with a modified suberin composition facilitated physiological studies revealing that the strong reduction in suberin in cyp86a1 mutants results in increased root water and solute permeabilities. The enhanced suberin 1 mutant, characterized by twofold increased root suberin content, has increased water-use efficiency and is affected in mineral ion uptake and transport. In this review the most recent findings on the biosynthesis and physiological importance of suberin in Arabidopsis are summarized and discussed.

Roots of terrestrial plants are designed to take up water and nutrients. At the same time, uptake of unwanted compounds, for example toxic, and infection by soil borne pathogens must be avoided. Specific unicellular tissues, the endodermis and the exodermis, allow roots to establish and maintain this selectivity. The endodermis represents an unicellular cell layer separating the central cylinder of the root from the cortex. The exodermis represents an unicellular cell layer located at the outer surface of the root directly below the root epidermis. Both tissues are characterised by specific cell wall modifications. In early developmental stages the anticlinal radial walls exhibit Casparian bands, composed of the polymers suberin and lignin. In a subsequent developmental state a suberin lamella is deposited on the inner surface of endo- and exodermal cell walls. These apoplastic barriers, mainly composed of suberin, significantly affect radial uptake of water and dissolved nutrients and radial loss of oxygen. Key Concepts: Casparian bands, composed of lignin and suberin, form characteristic cell wall modifications in radial walls of endodermal and exodermal cells. Suberin lamellae are deposited onto the inner surface of endodermal and exodermal cell walls. Apoplastic barriers in roots are established by the deposition of suberin into and onto the cell wall. Suberised apoplastic barriers in roots help to establish root selectivity in nutrient uptake. The endodermis, forming an ‘inner’ apoplastic barrier in the root, is important in preventing nutrients actively concentrated in the xylem from passively diffusing back to the soil. The exodermis, forming an ‘outer’ apoplastic barrier at the root surface, mainly establishes the root/soil interface between the root and the soil environment surrounding the root. As an adaptation to various environmental stress factors (e.g. drought, salt, heavy metal stress, oxygen deficiency, etc.) suberisation of apoplastic barriers is significantly modified. Fatty acid elongases and cytochrome P450 hydroxylases represent important key enzymes in suberin biosynthesis. Keywords: apoplastic barrier; Casparian band; endodermis; exodermis; hypodermis; nutrient uptake; plant root; suberin; transport

Floral organs display tremendous variation in their exterior that is essential for organogenesis and the interaction with the environment. This diversity in surface characteristics is largely dependent on the composition and structure of their coating cuticular layer. To date, mechanisms of flower organ initiation and identity have been studied extensively, while little is known regarding the regulation of flower organs surface formation, cuticle composition, and its developmental significance. Using a synthetic microRNA approach to simultaneously silence the three SHINE (SHN) clade members, we revealed that these transcription factors act redundantly to shape the surface and morphology of Arabidopsis flowers. It appears that SHNs regulate floral organs' epidermal cell elongation and decoration with nanoridges, particularly in petals. Reduced activity of SHN transcription factors results in floral organs' fusion and earlier abscission that is accompanied by a decrease in cutin load and modified cell wall properties. SHN transcription factors possess target genes within four cutin- and suberin-associated protein families including, CYP86A cytochrome P450s, fatty acyl-CoA reductases, GSDL-motif lipases, and BODYGUARD1-like proteins. The results suggest that alongside controlling cuticular lipids metabolism, SHNs act to modify the epidermis cell wall through altering pectin metabolism and structural proteins. We also provide evidence that surface formation in petals and other floral organs during their growth and elongation or in abscission and dehiscence through SHNs is partially mediated by gibberellin and the DELLA signaling cascade. This study therefore demonstrates the need for a defined composition and structure of the cuticle and cell wall in order to form the archetypal features of floral organs surfaces and control their cell-to-cell separation processes. Furthermore, it will promote future investigation into the relation between the regulation of organ surface patterning and the broader control of flower development and biological functions., Author Summary The cuticular layer that covers all aerial parts of plants plays a vital role not only in the interaction with environment but also in plant development and growth. Despite the recent significant achievements in the identification of structural genes involved in cuticle biosynthesis and secretion, little is known regarding the regulation of metabolic pathways generating cuticular constituents, more specifically wax and cutin. The Arabidopsis AP2-type transcription factor SHINE1/WAX INDUCER1 (SHN1/WIN1) was the first assigned regulator of a cuticle-related metabolic pathway; nevertheless, its mode of action and biological function remain uncertain due to redundancy with two additional clade members. Here, by co-silencing all three SHN clade members using an artificial microRNAs approach, we demonstrated that SHN transcription factors act redundantly in patterning reproductive organ surface, modulating processes associated with cell elongation, adhesion, and separation, which secure the proper function of these organs. It appears that SHN transcription factors act directly on downstream cutin and cell wall–modifying genes. These factors are likely part of the genetic network controlling floral organ development. Thus, SHN transcription factors link together cuticle assembly, cell wall remodeling, and flower development to form the archetypal surface of floral organs mediating plant reproduction through pollination and seed dispersal.

The elicitor-induced incorporation of phenylpropanoid derivatives into the cell wall and the secretion of soluble coumarin derivatives (phytoalexins) by parsley (Petroselinum crispum L.) suspension cultures can be potentiated by pretreatment of the cultures with 2,6-dichloroisonicotinic acid or derivatives of salicylic acid. To investigate this phenomenon further, the cell walls and an extracellular soluble polymer were isolated from control cells or cells treated with an elicitor from Phytophthora megasperma f. sp. glycinea. After alkaline hydrolysis, both fractions from elicited cells showed a greatly increased content of 4-coumaric, ferulic, and 4-hydroxybenzoic acid, as well as 4-hydroxybenzaldehyde and vanillin. Two minor peaks were identified as tyrosol and methoxytyrosol. The pretreatment effect is most pronounced at a low elicitor concentration. Its specificity was elaborated for coumarin secretion. When the parsley suspension cultures were preincubated for 1 d with 2,6-dichloroisonicotinic, 4- or 5-chlorosalicylic, or 3,5- dichlorosalicylic acid, the cells exhibited a greatly increased elicitor response. Pretreatment with isonicotinic, salicylic, acetylsalicylic, or 2,6-dihydroxybenzoic acid was less efficient in enhancing the response, and some other isomers were inactive. This increase in elicitor response was also observed for the above-mentioned monomeric phenolics, which were liberated from cell walls upon alkaline hydrolysis and for "lignin-like" cell wall polymers determined by the thioglycolic acid method. It was shown for 5-chlorosalicylic acid that conditioning most likely improves the signal transduction leading to the activation of genes encoding phenylalanine ammonia lyase and 4-coumarate: coenzyme A ligase. The conditioning thus sensitizes the parsley suspension cells to respond to lower elicitor concentrations. If a similar mechanism were to apply to whole plants treated with 2,6-dichloroisonicotinic acid, a known inducer of systemic acquired resistance, one can hypothesize that fungal pathogens might be recognized more readily and effectively.

Lipides acyle dans Arabidopsis et toutes les autres plantes ont une myriade de fonctions diverses. Il s'agit notamment de fournir la barriere de diffusion de base des membranes qui separe les cellules et les organites subcellulaires. Cette fonction implique seuls plus de 10 classes de lipides membranaires, y compris les phospholipides, galactolipides, et sphingolipides, et au sein de chaque classe, les variations dans la composition de la chaine acyle augmenter le nombre de structures a plusieurs centaines de possibles especes moleculaires. Lipides acyle dans le sous forme de compte triacylglycerol de 35% du poids de graines d'Arabidopsis et representent leur forme majeure de carbone et de stockage d'energie. Une couche de cutine et les cires cuticulaires qui limite la perte d'eau et offre une protection contre les invasions par des agents pathogenes et autres stress recouvre toute la surface aerienne d'Arabidopsis. Des fonctions similaires sont fournis par la suberine et des cires associes qui sont localisees dans les racines, les teguments et les zones d'abscission et sont produites en reponse a une blessure. Ce chapitre se concentre sur les voies metaboliques qui sont associes a la biosynthese et la degradation des lipides acyle mentionnes ci-dessus. Ces voies, les enzymes et les genes sont egalement presentes en detail dans un site Web associe (ARALIP: http://aralip.plantbiology.msu.edu/ ). Protocoles et methodes utilises pour l'analyse des lipides Arabidopsis sont fournis. Enfin, un resume detaille de la composition des lipides Arabidopsis est fourni en trois chiffres et 15 tables.

Epidermal cell layers play important roles in plant defenses against various environmental stresses. Here we report the identification of a cuticle membrane mutant, wilted dwarf and lethal 1 (wdl1), from a rice T-DNA insertional population. The mutant is dwarf and die at seedling stage due to increased rates of water loss. Stomatal cells and pavement cells are smaller in the mutant, suggesting that WDL1 affects epidermal cell differentiation. T-DNA was inserted into a gene that encodes a protein belonging to the SGNH subfamily, within the GDSL lipase superfamily. The WDL1-sGFP signal coincided with the RFP signal driven by AtBIP-mRFP, indicating that WDL1 is an ER protein. SEM analyses showed that their leaves have a disorganized crystal wax layer. Cross-sectioning reveals loose packing of the cuticle and irregular thickness of cell wall. Detailed analyses of the epicuticular wax showed no significant changes either in the total amount and amounts of each monomer or in the levels of lipid polymers, including cutin and other covalently bound lipids, attached to the cell wall. We propose that WDL1 is involved in cutin organization, affecting depolymerizable components.

Suberin and waxes embedded in the suberin polymer are key compounds in the control of transpiration in the tuber periderm of potato (Solanum tuberosum). Suberin is a cell-wall biopolymer with aliphatic and aromatic domains. The aliphatic suberin consists of a fatty acid polyester with esterified ferulic acid, which is thought to play an important role in cross-linking to the aromatic domain. In potato, ferulic acid esters are also the main components of periderm wax. How these ferulate esters contribute to the periderm water barrier remains unknown. Here we report on a potato gene encoding a fatty omega-hydroxyacid/fatty alcohol hydroxycinnamoyl transferase (FHT), and study its molecular and physiological relevance in the tuber periderm by means of a reverse genetic approach. In FHT RNAi periderm, the suberin and its associated wax contained much smaller amounts of ferulate esters, in agreement with the in vitro ability of the FHT enzyme to conjugate ferulic acid with omega-hydroxyacid and fatty alcohols. FHT down-regulation did not affect the typical suberin lamellar ultrastructure but had significant effects on the anatomy, sealing properties and maturation of the periderm. The tuber skin became thicker and russeted, water loss was greatly increased, and maturation was prevented. FHT deficiency also induced accumulation of the hydroxycinnamic acid amides feruloyl and caffeoyl putrescine in the periderm. We discuss these results in relation to the role attributed to ferulates in suberin molecular architecture and periderm impermeability.

The anther cuticle and microspore exine act as protective barriers for the male gametophyte and pollen grain, but relatively little is known about the mechanisms underlying the biosynthesis of the monomers of which they are composed. We report here the isolation and characterization of a rice (Oryza sativa) male sterile mutant, cyp704B2, which exhibits a swollen sporophytic tapetal layer, aborted pollen grains without detectable exine, and undeveloped anther cuticle. In addition, chemical composition analysis indicated that cutin monomers were hardly detectable in the cyp704B2 anthers. These defects are caused by a mutation in a cytochrome P450 family gene, CYP704B2. The CYP704B2 transcript is specifically detected in the tapetum and the microspore from stage 8 of anther development to stage 10. Heterologous expression of CYP704B2 in yeast demonstrated that CYP704B2 catalyzes the production of ω -hydroxylated fatty acids with 16 and 18 carbon chains. Our results provide insights into the biosynthesis of the two biopolymers sporopollenin and cutin. Specifically, our study indicates that the ω -hydroxylation pathway of fatty acids relying on this ancient CYP704B family, conserved from moss to angiosperms, is essential for the formation of both cuticle and exine during plant male reproductive and spore development.

Though central to our understanding of how roots perform their vital function of scavenging water and solutes from the soil, no direct genetic evidence currently exists to support the foundational model that suberin acts to form a chemical barrier limiting the extracellular, or apoplastic, transport of water and solutes in plant roots. Using the newly characterized enhanced suberin1 (esb1) mutant, we established a connection in Arabidopsis thaliana between suberin in the root and both water movement through the plant and solute accumulation in the shoot. Esb1 mutants, characterized by increased root suberin, were found to have reduced day time transpiration rates and increased water-use efficiency during their vegetative growth period. Furthermore, these changes in suberin and water transport were associated with decreases in the accumulation of Ca, Mn, and Zn and increases in the accumulation of Na, S, K, As, Se, and Mo in the shoot. Here, we present direct genetic evidence establishing that suberin in the roots plays a critical role in controlling both water and mineral ion uptake and transport to the leaves. The changes observed in the elemental accumulation in leaves are also interpreted as evidence that a significant component of the radial root transport of Ca, Mn, and Zn occurs in the apoplast., Author Summary The root system is a highly specialized plant organ that works to get both water and essential mineral nutrients from the changing chemically and physically complex environment of the soil. Roots do this by both controlling the uptake of water and essential mineral ions, as well as regulating their movement to the central vascular system of the plant for long distance transport to the shoot. To allow the cellular control of water and mineral ion uptake and transport via specialized transport proteins, plant roots contain a waxy layer of suberin that acts to seal connections between cells, preventing uncontrolled leakage of water and mineral ions between cells. By screening thousands of mutant A. thaliana plants, we were able to identify a plant with elevated levels of suberin in the root. Using this mutant, we were able to uncover the importance of suberin in sealing connections between root cells to regulate water movement through the plant and accumulation of various essential and nonessential minerals in leaves, including sodium, sulfur, potassium, calcium, manganese, zinc, arsenic, selenium, and molybdenum.

Increasing soil salinity reduces crop yields worldwide, with rice being particularly affected. We have examined the correlation between apoplastic barrier formation in roots, Na+ uptake into shoots and plant survival for three rice (Oryza sativa L.) cultivars of varying salt sensitivity: the salt-tolerant Pokkali, moderately tolerant Jaya and sensitive IR20. Rice plants grown hydroponically or in soil for 1 month were subjected to both severe and moderate salinity stress. Apoplastic barriers in roots were visualized using fluorescence microscopy and their chemical composition determined by gas chromatography and mass spectrometry. Na+ content was estimated by flame photometry. Suberization of apoplastic barriers in roots of Pokkali was the most extensive of the three cultivars, while Na+ accumulation in the shoots was the least. Saline stress induced the strengthening of these barriers in both sensitive and tolerant cultivars, with increase in mRNAs encoding suberin biosynthetic enzymes being detectable within 30 min of stress. Enhanced barriers were detected after several days of moderate stress. Overall, more extensive apoplastic barriers in roots correlated with reduced Na+ uptake and enhanced survival when challenged with high salinity.

The cuticle fulfills multiple roles in the plant life cycle, including protection from environmental stresses and the regulation of organ fusion. It is largely composed of cutin, which consists of C16-18 fatty acids. While cutin composition and biosynthesis have been studied, the export of cutin monomers out of the epidermis has remained elusive. Here, we show that DESPERADO (AtWBC11) (abbreviated DSO), encoding a plasma membrane-localized ATP-binding cassette transporter, is required for cutin transport to the extracellular matrix. The dso mutant exhibits an array of surface defects suggesting an abnormally functioning cuticle. This was accompanied by dramatic alterations in the levels of cutin monomers. Moreover, electron microscopy revealed unusual lipidic cytoplasmatic inclusions in epidermal cells, disappearance of the cuticle in postgenital fusion areas, and altered morphology of trichomes and pavement cells. We also found that DSO is induced by salt, abscisic acid, and wounding stresses and its loss of function results in plants that are highly susceptible to salt and display reduced root branching. Thus, DSO is not only essential for developmental plasticity but also plays a vital role in stress responses.

A fatty-acid-metabolizing enzyme from Arabidopsis thaliana, CYP94C1, belonging to the cytochrome P450 family was cloned and characterized. CYP94C1 was heterologously expressed in a Saccharomyces cerevisiae strain (WAT11) engineered for P450 expression. When recombinant yeast microsomes were incubated with lauric acid (C12:0) for 15 min, one major metabolite was formed. The product was purified and identified by GC/MS as 12-hydroxylauric acid. Longer incubation (40 min) led to the formation of an additional metabolite identified by GC/MS as dodecadioic acid. This diacid was also produced by incubation with 12-hydroxylauric acid. These compounds were not produced by incubating microsomes from yeast transformed with a void plasmid, demonstrating the involvement of CYP94C1. This new enzyme also metabolized fatty acids of varying aliphatic chain lengths (C12 to C18) and in-chain modifications, for example, degree of unsaturation or the presence of an epoxide as an additional polar functional group. Transcription of the gene encoding CYP94C1 is enhanced by stress, treatment with the hormone methyl jasmonate and wounding. Treatment with methyl jasmonate also induced lauric acid metabolism in microsomes prepared from Arabidopsis. The induction of hydroxylase activity was dose dependent and increased with exposure time, reaching 16x higher in microsomes from 24-h treated Arabidopsis compared with control plants. Analysis of the metabolites showed a mixture of 12-, 11- and 10-hydroxylauric acids, revealing for the first time the presence of fatty acid in-chain hydroxylase in Arabidopsis.