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Suberin — a biopolyester forming apoplastic plant interfaces
Suberin — a biopolyester forming apoplastic plant interfaces Rochus Franke and Lukas Schreiber 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. Addresses Institute of Cellular and Molecular Botany, University of Bonn, Kirschallee 1, D-53115 Bonn, Germany Corresponding author: Franke, Rochus ([email protected])
Occurrence and function in plants The main function of the lipophilic polyester is the formation of an interface separating either the living plant tissue from the environment or different tissues within the plant body. Thereby, suberin depositions significantly reduce uncontrolled transport of water, dissolved ions (e.g. nutrients and toxins) and gases. The tissue distribution of suberized cell walls in plants suggests that suberization occurs wherever and whenever a plant needs to form a barrier [1]. The suberized periderms of shoots and roots establish an interface between the plant body and the environment. This is best known for cork, the heavily suberized (>50% w/w) outer bark of the cork oak (Quercus suber) [6]. The periderm of storage organs, such as potato (Solanum tuberosum) tubers, also has a high suberin content [7,8]. During wound healing, healthy tissue is protected by suberin depositions (wound periderm) at the wound edges [1]. In Citrus seed, suberin depositions in the chalaza micropyle region of the seed coat seal off the seed after disconnection from the vascular tissue [2]. Suberin depositions in the bundle sheath cell walls of C4 plants separate mesophyll cells from vascular tissue [2].
Current Opinion in Plant Biology 2007, 10:252–259 This review comes from a themed issue on Physiology and metabolism Edited by Clint Chapple and Malcolm M Campbell Available online 16th April 2007 1369-5266/$ – see front matter # 2007 Elsevier Ltd. All rights reserved. DOI 10.1016/j.pbi.2007.04.004
Introduction When plants colonized the terrestrial environment, they had to protect themselves against desiccation. One strategy was the formation of suberin, an extracellular biopolymer. Suberin consists of a polyaliphatic domain, typically in close association with a polyaromatic domain that is derived from ferulic acid [1–3]. The function of suberin as a transport barrier for water and solutes is, however, mostly determined by the aliphatic domain [4,5]. Therefore, the chemical composition and physiological properties of aliphatic suberin have been the focus of recent suberin research. This review summarizes recent developments in our understanding of the structure and biosynthesis of aliphatic suberin, and its physiological importance as an apoplastic transport barrier. Current Opinion in Plant Biology 2007, 10:252–259
In primary roots, suberin depositions occur at two distinct layers: in endodermal cell walls (ECW), separating the vascular tissue of the central cylinder from the root cortex, and in rhizodermal and hypodermal cell walls (RHCW), building the interface to the soil environment [9]. Thereby, suberized barriers in roots influence the radial transport of solutes and dissolved ions in the apoplast, and function in water and nutrient uptake and protection against pathogens and toxic compounds in the rhizophere [4,5]. Histochemical and microscopical methods revealed that plants respond to environmental stimuli, such as drought, salt stress or oxygen deficiency, with increased suberization of apoplastic barriers in roots [10–12].
The chemical composition and macromolecular structure model Our current knowledge about suberin structure derives mainly from compositional data generated by depolymerization and subsequent analysis of the depolymerization products using gas chromatography coupled to mass spectrometry (GC–MS). Aliphatic suberin is a biopolyester primarily composed of oxygenated fatty acid derivatives. The main monomers released by transesterification are v-hydroxyacids (v-OH-acids) and a,v-dicarboxylic acids (a,v-diacids), ranging in chain length from C16 to C30, and glycerol (Table 1; [6,13]). Alcohols and unsubstituted fatty acids are minor components present in suberin. Although this substance class composition www.sciencedirect.com
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Table 1 Suberin composition. Major monomers (exemplified as C18 chain-length molecules) and di- and oligomeric building blocks obtained by suberin depolymerization.
1. 18-Hydroxy-octadecenoic acid esterified to 18-hydroxy-octadecenoic acid. 2. 9-Epoxy-18-hydroxy-octadecanoic acid esterified to 9-epoxyoctadecane-1,18-dioic acid. 3. Glycerol esterified to octadecene-1,18-dioic acid. 4. Glycerol esterified to 9-Epoxy-octadecane-1,18-dioic acid esterified to glycerol. 5. 9-Epoxy-octadecane-1,18-dioic acid esterified to glycerol esterified to octadecene-1,18-dioic acid. 6. Ferulic acid esterified to 22-hydroxy-docosanoic acid esterified to glycerol.
of suberin seems to be general for most suberin sources, some species-dependent variation in chain-length distribution and in-chain modification exist. Among the suberin model species, suberin from Q. suber bark is characterized by in-chain epoxides and vicinal diols [6], whereas suberin from potato tuber periderm [8,14] www.sciencedirect.com
and Arabidopsis roots [15] is characterized by monounsaturated v-OH-acids and a,v-diacids. Recently developed methods, such as partial hydrolysis and electrospray ionization coupled with tandem mass spectrometry, provided the first insights into the linkages within the suberin polymer. Linear dimers of v-OH-acids, Current Opinion in Plant Biology 2007, 10:252–259
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esterified to a,v-diacids, and interlinked v-OH-acids have been identified after partial suberin hydrolysis using calcium hydroxide (Table 1; [16]). An array of glyceryl esters identified by the same techniques determined glycerol esterified to v-OH-acids or a,v-diacids, esterified to both ends of an a,v-diacid, and as a linker between two a,v-diacids [17]. Consistent with recent macromolecular structure models of suberin [1,3], this set of building blocks was completed by the structural characterization of a trimeric diester of glycerol linked to a v-OH-acid linked to ferulic acid [18]. From this structure, it was proposed that ferulic acid covalently links the aliphatic suberin polyester to the adjacent polyaromatic domain. Building on the model postulated by Kolattukudy and co-workers some 30 years ago [19], the tentative macromolecular structure derived from the monomeric and oligomeric building blocks contains long-chain a,v-diacids esterified to glycerol at both ends as the core of the suberin macromolecule. From the polyol glycerol, the suberin polymer grows two- and three-dimensionally by the formation of ester linkages to additional a,v-diacids and vOH-acids. At the ‘edge’ of the glycerol-based polymer, ferulic acid attaches the aliphatic polyester to the polyaromatics, which themselves are thought to form the link to the cell wall carbohydrates.
Suberin forms a physiologically important transport barrier In contrast to the compositional similarity among species, the quantity of apoplastic suberization is strongly species dependent. The introduction of GC–MS techniques for the characterization of suberized cell walls in roots [20] revealed that absolute amounts of suberin measured in the RHCW and ECW of several species varied by more than two orders of magnitude [14]. From this large variation in root cell wall suberization, we can expect large differences in the efficiency of the cell walls as apoplastic transport barriers. Root suberization also varies quantitatively upon environmental conditions, indicating physiological relevance [4,5,21]. Analytical methods, combined with an experimental setup to determine the hydraulic conductivity in roots (using pressure probes [22]), revealed a correlation between hydraulic conductivity and the extent of suberization in the root apoplast [4]. Maize (Zea mays) roots grown in humid air had a suberin content in the RHCW 2.4 times greater than that of roots grown in aerated nutrient solution. As a result, the radial apoplastic water transport of humid-grown roots decreased substantially, by a factor of 1.5–3.5 [4]. On the basis of these experiments and similar studies [5], it was hypothesized that plants adjust the degree of suberization in their tissues in response to environmental stimuli, thereby regulating the apoplastic transport of water and solutes. Current Opinion in Plant Biology 2007, 10:252–259
The barrier properties of suberized cell walls in roots need to meet various demands. The plant root is designed to take up water and nutrients. At the same time, it should not take up toxic compounds and should prevent desiccation (water loss) under drought conditions. The roots of wetland plants in flooded soils are exposed to toxic compounds that are derived from anoxic microbial activity. Also, reduced ions need to be excluded and oxygen loss needs to be minimized. Anatomical, chemical and microelectrode studies on roots of Amazonian floodplain species revealed a correlation between the suberin content in the RHCW and the ability to prevent radial oxygen loss (ROL) [23]. When microelectrodes were used to measure the oxygen concentration in individual root tissues of the wetland species Phragmites australis and Glyceria maxima, the suberized tissue of the exodermis was found to be the barrier that restricts ROL [24]. In addition, anatomical studies revealed that periodic acid could not penetrate the suberized hypodermal cell layers of Phragmites roots, whereas the lignified cell walls did not provide a barrier for periodic acid diffusion. This can also be assumed for smaller molecules such as oxygen. Quantitative analytical methods were used to show that the suberization of apoplastic barriers in the roots of Ricinus communis changes in response to salt stress [21]. In the presence of 100 mM NaCl, the aliphatic suberin content increased significantly in ECW and RHCW. Under salt stress, plants reinforce apoplastic barriers to prevent NaCl uptake in toxic concentrations. Similarly, maize roots responded to salt stress by substantially increasing suberization in ECW [25]. These studies, which combined quantitative analytical data with physiological measurements, showed that suberin restricts the diffusion of water, solutes and gases. However, a complete blockage of transport across the apoplastic barrier was never observed. Thus, a question arises: to what extent does suberin accumulation and composition determine barrier properties. The rice endodermis contains 35 times more suberin than that of maize, but the hydraulic conductivity in maize roots was only three times higher than that in rice roots [26]. Apoplastic water transport in rice could be reduced threeto four-fold by the deposition of copper-ferrocyanide precipitates in the root exodermis, indicating the occurrence of a partially porous structure in these suberized cell walls [27]. Similarly, the wound periderm of potato had water permeabilities that were about 100-fold higher than the water permeabilities of native periderm, although the wound periderm contained about 60% of the native suberin quantity [14]. Deeper insights into the factors that determine the properties of suberin can be expected from plants in which the biosynthesis, three-dimensional structure and topographical deposition of suberin are affected.
Biosynthesis and deposition of suberin Despite the physiological importance of suberin, its biosynthesis and deposition remain enigmatic. A hypothetical www.sciencedirect.com
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pathway has been postulated in Arabidopsis [15] on the basis of the detailed compositional data available, anticipating the enzyme activities required for the synthesis of suberin precursors (Figure 1). Diagnostic for suberin are
very long chain derivatives (>C18–C30) and a,v-diacids [1,28]. Therefore, fatty acid elongation and v-carbon oxidation represent two characteristic processes of suberin biosynthesis. This is supported by recent metabolite
Figure 1
Simplified model of the suberin biosynthetic pathway (adapted from [15]). The building blocks for suberin are glycerol and C16 and C18 fatty acids derived from primary metabolic activity, and ferulic acid, a product of the phenylpropanoid pathway. Depending on the species, fatty acid suberin precursors undergo mid-chain modifications, resulting in unsaturated or epoxy fatty acids. Saturated fatty acids can be elongated up to C30 suberin precursor. The majority of the suberin precursors enter a v-oxidation pathway, resulting in v-hydroxyacids and a,v-diacids. The vhydroxyacids and a,v-diacids form esters by linkage to glycerol and/or ferulic acid or are interlinked. After export to the apoplast, the mono- and oligomeric building blocks will be polymerized into the suberin macromolecule. The deduced catalysts that are typically responsible for the diagrammed reactions are shown. ABC, ATP binding cassette transporter; AT, acyltransferase; FAD, fatty acid desaturase; FAE, fatty acid elongase complex; KCS, b-ketoacyl-CoA synthases; hDH, v-hydroxyacid dehydrogenase; HDL, a,b-hydrolase; oDH, v-oxoacid dehydrogenase; P450, cytochrome P450 monooxygenase; PA, polyaromatic domain; PM, plasma membrane; POd, peroxidase; POg, peroxygenase. www.sciencedirect.com
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profiling studies in wound healing potato tubers in which v-oxidation and elongation were found to be the main metabolic fates of radiolabeled fatty acid precursors during suberization [29].
tobacco P450, CYP94A5, also catalyzes the oxidation of v-OH-acids to a,v-diacids, in addition to the preceding v-hydroxylation, indicating that this multi-step reaction can be catalyzed by a single enzyme [41].
In plants, acyl elongation is catalyzed by the fatty acid elongation complex (FAE), which consists of four enzymatic subunits [30]. The b-ketoacyl-CoA synthases (KCS), which catalyze the initial step in FAE synthesis, were demonstrated to be rate limiting and to determine the chain length of the FAE products [31]. To date, no kcs mutants with phenotypes that are related to suberin have been described, but mutant phenotypes have been described for cutin and wax [30,32]. Recently, a FAE activity in microsomal preparations of maize primary roots has been shown to elongate C18 acyl-CoA to C24 [33]. These products and the substrate preference for C18 and C20 acyl-CoA correlated with the chain-length distribution of suberin in maize root seedlings. Similarly, systematic approaches in which all Arabidopsis KCS were heterologously expressed in yeast revealed activities suitable for the synthesis of Arabidopsis suberin precursors [34,35]. The KCS enzymes encoded by At1g04220 and At5g43760 elongated C18 and C20 precursors, whereas the enzyme encoded by At2g16280 elongated C18–C22 substrates. The products of these three KCS enzymes had chain-length of C22 and C24, which are characteristic of Arabidopsis root suberin. These biochemical studies do not, however, provide direct evidence for the involvement of any of the KCS in suberin biosynthesis, because elongated fatty acids are also required as precursors in other biosynthetic pathways.
Depending on the species, monomers with other fatty acid modifications can be part of the suberin polymer. The mid-chain oxygenation, as found in monomers of Q. suber suberin, is introduced either by a P450 and/or by a peroxygenase-dependent pathway [1,37]. On the basis of the content of unsaturated C18 aliphatics in Arabidopsis and potato suberin, it has been proposed that fatty acid desaturases (FAD) are involved in the biosynthesis of 30–40% of the suberin monomers [8,15]. Some of these and subsequent reactions presumably involve activated (e.g. CoA) fatty acid derivatives [1,30]; therefore, long-chain acyl CoA synthethase (LACS) and a thioesterase to release the CoA are required. Interestingly, recombinant LACS2, which was shown to affect cuticle formation, had a substrate preference for v-hydroxy acids [46]. The non-aliphatic suberin constituent glycerol derives from C3 metabolites in primary metabolism and the ferulic acid is provided by the phenylpropanoid pathway [47].
Hydroxylation of fatty acids in the v-position is typically catalyzed by NADPH-dependent cytochrome P450 monooxygenases (P450), particularly of the CYP86 and CYP94 families [36,37]. The genes encoding CYP86A1 (Arabidopsis) [38], CYP94A1 (Vicia sativa) [39], CYP94A2 (V. sativa) [40], and CYP94A5 (Nicotiana tabacum) [41] have been cloned on the basis of sequence similarity, and the enzymes were subsequently biochemically characterized as v-hydroxylases after heterologous expression in yeast. Arabidopsis mutants in CYP86A8 [42] and CYP86A2 [43] are characterized by phenotypes involving defective cuticle formation, but the suberin of the mutants was not analyzed. Hence, it is still a matter of debate whether any of these v-hydroxylases are involved in suberin biosynthesis or not. The formation of a,v-diacids from v-OH-acids was demonstrated in suberizing tissue almost 30 years ago [44]. This dehydrogenase activity, which has been characterized and partially purified from suberizing potato tuber discs, was shown to be a two-step mechanism involving a v-oxo acid as intermediate (reviewed in [1]). The first enzyme might be encoded by HOTHEAD-like oxidoreductase genes, as indicated by the reduced amounts of a,v-diacids in the cutin of the hothead mutant [45]. However, the Current Opinion in Plant Biology 2007, 10:252–259
Our current knowledge of how these monomers are linked together, transferred to the apoplast and incorporated into the suberin polyester is insufficient. Glycerol- and ferulic-acid-dependent acyltransferases are required to form the oligomeric building blocks described above. In Arabidopsis, there are at least 19 potential acyltransferases that have been identified through sequence similarity [48]. Traditionally, building units for cell wall polymers were thought to be exported by a vesicular pathway from the endoplasmic reticulum to their extracellular destination. Recently, CER5, encoding an ABCtransporter, was identified as a factor in the transfer of aliphatic wax precursor across the plasma membrane [49]. Whether a direct export of aliphatic suberin precursor also occurs via an ABC-transporter, as hypothesized for longchain aliphatic wax precursors, remains to be elucidated. As deduced from cuticular defects in a mutant affected in the extracellular protein BODYGUARD, the formation of the suberin polymer by esterification probably involves members of the a,b-hydrolase superfamily, which contains hydrolases, esterases, cutinases and lipases [50]. Finally, the aliphatic polyester is linked to the polyaromatic domain via ferulic acid, presumably in a step catalyzed by cell wall peroxidases [51,52]. Suberin deposition occurs only in specific tissues and, in the case of Casparian bands, even at a specific site in the radial cell wall of a single cell. This and the induction of suberization in response to environmental conditions indicate a complex regulation of suberin biosynthesis, for which the molecular mechanisms are totally unknown to date. www.sciencedirect.com
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parsnip, rutabaga, turnip, red beet and sweet potato by combined gas-liquid chromatography and mass spectrometry. Plant Physiol 1975, 55:567-573.
Conclusions There is still a great gap in our knowledge of suberin biosynthesis. The core reactions in suberin biosynthesis, catalyzed by P450 and FAE complexes, take place at the endoplasmic reticulum membranes. The membraneassociation in complexes and the lack of commercially available substrates (e.g. long chain v-OH-acid-CoA) handicaps the progress of biochemical studies favoring molecular genetic approaches. The hypothetical pathway of suberin biosynthesis (Figure 1), which is based on knowledge of the chemical composition of Arabidopsis suberin [15] combined with a list of candidate genes for lipid metabolism [48], will lead to the identification of suberin mutants in future forward and reverse genetic approaches. This process will be supported by information available from tissue-specific root expression of candidate genes from genome [53] and family-focused (P450 [54]) microarray studies. The identification of the molecular elements of suberin formation might enable suberin engineering strategies to improve resistance to environmental stress conditions in agriculturally important plants. The unique properties of cork combine low permeability, low temperature conductivity, and high chemical resistance with high elasticity and low weight. These properties and cork’s chemical composition, which is dominated by long-chain aliphates with terminal functional groups, have drawn attention to the use of suberin biosynthetic products in industrial applications. These include the use of cork products in composite materials as sealant or insulators and the utility of plant-produced suberin monomers as bio-based lubricants or polyester foams [55].
References and recommended reading Papers of particular interest, published within the period of review, have been highlighted as: of special interest of outstanding interest 1.
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46. Schnurr J, Shockey J, Browse J: The acyl-CoA synthetase encoded by LACS2 is essential for normal cuticle development in Arabidopsis. Plant Cell 2004, 16:629-642.
32. Nawrath C: Unraveling the complex network of cuticular structure and function. Curr Opin Plant Biol 2006, 9:281-287.
47. Boerjan W, Ralph J, Baucher M: Lignin biosynthesis. Annu Rev Plant Biol 2003, 54:519-546.
33. Schreiber L, Franke R, Lessire R: Biochemical characterization of elongase activity in corn (Zea mays L.) roots. Phytochemistry 2005, 66:131-138. Using microsomal preparation from corn roots, the authors showed that fatty acid elongation activity is associated with suberized tissue.
48. Beisson F, Koo AJK, Ruuska S, Schwender J, Pollard M, Thelen JJ, Paddock T, Salas JJ, Savage L, Milcamps A et al.: Arabidopsis genes involved in acyl lipid metabolism. A 2003 census of the candidates, a study of the distribution of expressed sequence tags in organs, and a Web-based database. Plant Physiol 2003, 132:681-697.
34. Trenkamp S, Martin W, Tietjen K: Specific and differential inhibition of very-long-chain fatty acid elongases from Arabidopsis thaliana by different herbicides. Proc Natl Acad Sci USA 2004, 101:11903-11908. Current Opinion in Plant Biology 2007, 10:252–259
49. Pighin JA, Zheng H, Balakshin LJ, Goodman IP, Western TL, Jetter R, Kunst L, Samuels AL: Plant cuticular lipid export requires an ABC transporter. Science 2004, 306:702-704. www.sciencedirect.com
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50. Kurdyukov S, Faust A, Nawrath C, Ba¨r S, Voisin D, Efremova N, Franke R, Schreiber L, Saedler H, Me´traux JP, Yephremov A: The epidermis-specific extracellular BODYGUARD controls cuticle development and morphogenesis in Arabidopsis. Plant Cell 2006, 18:321-339. 51. Bernards MA, Summerhurst DK, Razem FA: Oxidases, peroxidases and hydrogen peroxide: the suberin connection. Phytochem Rev 2004, 3:113-126. 52. Arrieta-Baez D, Stark RE: Modeling suberization with peroxidase-catalyzed polymerization of hydroxycinnamic
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acids: cross-coupling and dimerization reactions. Phytochemistry 2006, 67:743-753. 53. Birnbaum K, Shasha DE, Wang JY, Jung JW, Lambert GM, Galbraith DW, Benfey PN: A gene expression map of the Arabidopsis root. Science 2003, 302:1956-1960. 54. Schuler MA, Werck-Reichhart D: Functional genomics of P450s. Annu Rev Plant Biol 2003, 54:629-667. 55. Gandini A, Neto CP, Silvestre AJD: Suberin: a promising renewable resource for novel macromolecular materials. Prog Polym Sci 2006, 31:878-892.
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Occurrence and function in plants The main function of the lipophilic polyester is the formation of an interface separating either the living plant tissue from the environment or different tissues within the plant body. Thereby, suberin depositions significantly reduce uncontrolled transport of water, dissolved ions (e.g. nutrients and toxins) and gases. The tissue distribution of suberized cell walls in plants suggests that suberization occurs wherever and whenever a plant needs to form a barrier [1]. The suberized periderms of shoots and roots establish an interface between the plant body and the environment. This is best known for cork, the heavily suberized (>50% w/w) outer bark of the cork oak (Quercus suber) [6]. The periderm of storage organs, such as potato (Solanum tuberosum) tubers, also has a high suberin content [7,8]. During wound healing, healthy tissue is protected by suberin depositions (wound periderm) at the wound edges [1]. In Citrus seed, suberin depositions in the chalaza micropyle region of the seed coat seal off the seed after disconnection from the vascular tissue [2]. Suberin depositions in the bundle sheath cell walls of C4 plants separate mesophyll cells from vascular tissue [2].
Current Opinion in Plant Biology 2007, 10:252–259 This review comes from a themed issue on Physiology and metabolism Edited by Clint Chapple and Malcolm M Campbell Available online 16th April 2007 1369-5266/$ – see front matter # 2007 Elsevier Ltd. All rights reserved. DOI 10.1016/j.pbi.2007.04.004
Introduction When plants colonized the terrestrial environment, they had to protect themselves against desiccation. One strategy was the formation of suberin, an extracellular biopolymer. Suberin consists of a polyaliphatic domain, typically in close association with a polyaromatic domain that is derived from ferulic acid [1–3]. The function of suberin as a transport barrier for water and solutes is, however, mostly determined by the aliphatic domain [4,5]. Therefore, the chemical composition and physiological properties of aliphatic suberin have been the focus of recent suberin research. This review summarizes recent developments in our understanding of the structure and biosynthesis of aliphatic suberin, and its physiological importance as an apoplastic transport barrier. Current Opinion in Plant Biology 2007, 10:252–259
In primary roots, suberin depositions occur at two distinct layers: in endodermal cell walls (ECW), separating the vascular tissue of the central cylinder from the root cortex, and in rhizodermal and hypodermal cell walls (RHCW), building the interface to the soil environment [9]. Thereby, suberized barriers in roots influence the radial transport of solutes and dissolved ions in the apoplast, and function in water and nutrient uptake and protection against pathogens and toxic compounds in the rhizophere [4,5]. Histochemical and microscopical methods revealed that plants respond to environmental stimuli, such as drought, salt stress or oxygen deficiency, with increased suberization of apoplastic barriers in roots [10–12].
The chemical composition and macromolecular structure model Our current knowledge about suberin structure derives mainly from compositional data generated by depolymerization and subsequent analysis of the depolymerization products using gas chromatography coupled to mass spectrometry (GC–MS). Aliphatic suberin is a biopolyester primarily composed of oxygenated fatty acid derivatives. The main monomers released by transesterification are v-hydroxyacids (v-OH-acids) and a,v-dicarboxylic acids (a,v-diacids), ranging in chain length from C16 to C30, and glycerol (Table 1; [6,13]). Alcohols and unsubstituted fatty acids are minor components present in suberin. Although this substance class composition www.sciencedirect.com
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Table 1 Suberin composition. Major monomers (exemplified as C18 chain-length molecules) and di- and oligomeric building blocks obtained by suberin depolymerization.
1. 18-Hydroxy-octadecenoic acid esterified to 18-hydroxy-octadecenoic acid. 2. 9-Epoxy-18-hydroxy-octadecanoic acid esterified to 9-epoxyoctadecane-1,18-dioic acid. 3. Glycerol esterified to octadecene-1,18-dioic acid. 4. Glycerol esterified to 9-Epoxy-octadecane-1,18-dioic acid esterified to glycerol. 5. 9-Epoxy-octadecane-1,18-dioic acid esterified to glycerol esterified to octadecene-1,18-dioic acid. 6. Ferulic acid esterified to 22-hydroxy-docosanoic acid esterified to glycerol.
of suberin seems to be general for most suberin sources, some species-dependent variation in chain-length distribution and in-chain modification exist. Among the suberin model species, suberin from Q. suber bark is characterized by in-chain epoxides and vicinal diols [6], whereas suberin from potato tuber periderm [8,14] www.sciencedirect.com
and Arabidopsis roots [15] is characterized by monounsaturated v-OH-acids and a,v-diacids. Recently developed methods, such as partial hydrolysis and electrospray ionization coupled with tandem mass spectrometry, provided the first insights into the linkages within the suberin polymer. Linear dimers of v-OH-acids, Current Opinion in Plant Biology 2007, 10:252–259
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esterified to a,v-diacids, and interlinked v-OH-acids have been identified after partial suberin hydrolysis using calcium hydroxide (Table 1; [16]). An array of glyceryl esters identified by the same techniques determined glycerol esterified to v-OH-acids or a,v-diacids, esterified to both ends of an a,v-diacid, and as a linker between two a,v-diacids [17]. Consistent with recent macromolecular structure models of suberin [1,3], this set of building blocks was completed by the structural characterization of a trimeric diester of glycerol linked to a v-OH-acid linked to ferulic acid [18]. From this structure, it was proposed that ferulic acid covalently links the aliphatic suberin polyester to the adjacent polyaromatic domain. Building on the model postulated by Kolattukudy and co-workers some 30 years ago [19], the tentative macromolecular structure derived from the monomeric and oligomeric building blocks contains long-chain a,v-diacids esterified to glycerol at both ends as the core of the suberin macromolecule. From the polyol glycerol, the suberin polymer grows two- and three-dimensionally by the formation of ester linkages to additional a,v-diacids and vOH-acids. At the ‘edge’ of the glycerol-based polymer, ferulic acid attaches the aliphatic polyester to the polyaromatics, which themselves are thought to form the link to the cell wall carbohydrates.
Suberin forms a physiologically important transport barrier In contrast to the compositional similarity among species, the quantity of apoplastic suberization is strongly species dependent. The introduction of GC–MS techniques for the characterization of suberized cell walls in roots [20] revealed that absolute amounts of suberin measured in the RHCW and ECW of several species varied by more than two orders of magnitude [14]. From this large variation in root cell wall suberization, we can expect large differences in the efficiency of the cell walls as apoplastic transport barriers. Root suberization also varies quantitatively upon environmental conditions, indicating physiological relevance [4,5,21]. Analytical methods, combined with an experimental setup to determine the hydraulic conductivity in roots (using pressure probes [22]), revealed a correlation between hydraulic conductivity and the extent of suberization in the root apoplast [4]. Maize (Zea mays) roots grown in humid air had a suberin content in the RHCW 2.4 times greater than that of roots grown in aerated nutrient solution. As a result, the radial apoplastic water transport of humid-grown roots decreased substantially, by a factor of 1.5–3.5 [4]. On the basis of these experiments and similar studies [5], it was hypothesized that plants adjust the degree of suberization in their tissues in response to environmental stimuli, thereby regulating the apoplastic transport of water and solutes. Current Opinion in Plant Biology 2007, 10:252–259
The barrier properties of suberized cell walls in roots need to meet various demands. The plant root is designed to take up water and nutrients. At the same time, it should not take up toxic compounds and should prevent desiccation (water loss) under drought conditions. The roots of wetland plants in flooded soils are exposed to toxic compounds that are derived from anoxic microbial activity. Also, reduced ions need to be excluded and oxygen loss needs to be minimized. Anatomical, chemical and microelectrode studies on roots of Amazonian floodplain species revealed a correlation between the suberin content in the RHCW and the ability to prevent radial oxygen loss (ROL) [23]. When microelectrodes were used to measure the oxygen concentration in individual root tissues of the wetland species Phragmites australis and Glyceria maxima, the suberized tissue of the exodermis was found to be the barrier that restricts ROL [24]. In addition, anatomical studies revealed that periodic acid could not penetrate the suberized hypodermal cell layers of Phragmites roots, whereas the lignified cell walls did not provide a barrier for periodic acid diffusion. This can also be assumed for smaller molecules such as oxygen. Quantitative analytical methods were used to show that the suberization of apoplastic barriers in the roots of Ricinus communis changes in response to salt stress [21]. In the presence of 100 mM NaCl, the aliphatic suberin content increased significantly in ECW and RHCW. Under salt stress, plants reinforce apoplastic barriers to prevent NaCl uptake in toxic concentrations. Similarly, maize roots responded to salt stress by substantially increasing suberization in ECW [25]. These studies, which combined quantitative analytical data with physiological measurements, showed that suberin restricts the diffusion of water, solutes and gases. However, a complete blockage of transport across the apoplastic barrier was never observed. Thus, a question arises: to what extent does suberin accumulation and composition determine barrier properties. The rice endodermis contains 35 times more suberin than that of maize, but the hydraulic conductivity in maize roots was only three times higher than that in rice roots [26]. Apoplastic water transport in rice could be reduced threeto four-fold by the deposition of copper-ferrocyanide precipitates in the root exodermis, indicating the occurrence of a partially porous structure in these suberized cell walls [27]. Similarly, the wound periderm of potato had water permeabilities that were about 100-fold higher than the water permeabilities of native periderm, although the wound periderm contained about 60% of the native suberin quantity [14]. Deeper insights into the factors that determine the properties of suberin can be expected from plants in which the biosynthesis, three-dimensional structure and topographical deposition of suberin are affected.
Biosynthesis and deposition of suberin Despite the physiological importance of suberin, its biosynthesis and deposition remain enigmatic. A hypothetical www.sciencedirect.com
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pathway has been postulated in Arabidopsis [15] on the basis of the detailed compositional data available, anticipating the enzyme activities required for the synthesis of suberin precursors (Figure 1). Diagnostic for suberin are
very long chain derivatives (>C18–C30) and a,v-diacids [1,28]. Therefore, fatty acid elongation and v-carbon oxidation represent two characteristic processes of suberin biosynthesis. This is supported by recent metabolite
Figure 1
Simplified model of the suberin biosynthetic pathway (adapted from [15]). The building blocks for suberin are glycerol and C16 and C18 fatty acids derived from primary metabolic activity, and ferulic acid, a product of the phenylpropanoid pathway. Depending on the species, fatty acid suberin precursors undergo mid-chain modifications, resulting in unsaturated or epoxy fatty acids. Saturated fatty acids can be elongated up to C30 suberin precursor. The majority of the suberin precursors enter a v-oxidation pathway, resulting in v-hydroxyacids and a,v-diacids. The vhydroxyacids and a,v-diacids form esters by linkage to glycerol and/or ferulic acid or are interlinked. After export to the apoplast, the mono- and oligomeric building blocks will be polymerized into the suberin macromolecule. The deduced catalysts that are typically responsible for the diagrammed reactions are shown. ABC, ATP binding cassette transporter; AT, acyltransferase; FAD, fatty acid desaturase; FAE, fatty acid elongase complex; KCS, b-ketoacyl-CoA synthases; hDH, v-hydroxyacid dehydrogenase; HDL, a,b-hydrolase; oDH, v-oxoacid dehydrogenase; P450, cytochrome P450 monooxygenase; PA, polyaromatic domain; PM, plasma membrane; POd, peroxidase; POg, peroxygenase. www.sciencedirect.com
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profiling studies in wound healing potato tubers in which v-oxidation and elongation were found to be the main metabolic fates of radiolabeled fatty acid precursors during suberization [29].
tobacco P450, CYP94A5, also catalyzes the oxidation of v-OH-acids to a,v-diacids, in addition to the preceding v-hydroxylation, indicating that this multi-step reaction can be catalyzed by a single enzyme [41].
In plants, acyl elongation is catalyzed by the fatty acid elongation complex (FAE), which consists of four enzymatic subunits [30]. The b-ketoacyl-CoA synthases (KCS), which catalyze the initial step in FAE synthesis, were demonstrated to be rate limiting and to determine the chain length of the FAE products [31]. To date, no kcs mutants with phenotypes that are related to suberin have been described, but mutant phenotypes have been described for cutin and wax [30,32]. Recently, a FAE activity in microsomal preparations of maize primary roots has been shown to elongate C18 acyl-CoA to C24 [33]. These products and the substrate preference for C18 and C20 acyl-CoA correlated with the chain-length distribution of suberin in maize root seedlings. Similarly, systematic approaches in which all Arabidopsis KCS were heterologously expressed in yeast revealed activities suitable for the synthesis of Arabidopsis suberin precursors [34,35]. The KCS enzymes encoded by At1g04220 and At5g43760 elongated C18 and C20 precursors, whereas the enzyme encoded by At2g16280 elongated C18–C22 substrates. The products of these three KCS enzymes had chain-length of C22 and C24, which are characteristic of Arabidopsis root suberin. These biochemical studies do not, however, provide direct evidence for the involvement of any of the KCS in suberin biosynthesis, because elongated fatty acids are also required as precursors in other biosynthetic pathways.
Depending on the species, monomers with other fatty acid modifications can be part of the suberin polymer. The mid-chain oxygenation, as found in monomers of Q. suber suberin, is introduced either by a P450 and/or by a peroxygenase-dependent pathway [1,37]. On the basis of the content of unsaturated C18 aliphatics in Arabidopsis and potato suberin, it has been proposed that fatty acid desaturases (FAD) are involved in the biosynthesis of 30–40% of the suberin monomers [8,15]. Some of these and subsequent reactions presumably involve activated (e.g. CoA) fatty acid derivatives [1,30]; therefore, long-chain acyl CoA synthethase (LACS) and a thioesterase to release the CoA are required. Interestingly, recombinant LACS2, which was shown to affect cuticle formation, had a substrate preference for v-hydroxy acids [46]. The non-aliphatic suberin constituent glycerol derives from C3 metabolites in primary metabolism and the ferulic acid is provided by the phenylpropanoid pathway [47].
Hydroxylation of fatty acids in the v-position is typically catalyzed by NADPH-dependent cytochrome P450 monooxygenases (P450), particularly of the CYP86 and CYP94 families [36,37]. The genes encoding CYP86A1 (Arabidopsis) [38], CYP94A1 (Vicia sativa) [39], CYP94A2 (V. sativa) [40], and CYP94A5 (Nicotiana tabacum) [41] have been cloned on the basis of sequence similarity, and the enzymes were subsequently biochemically characterized as v-hydroxylases after heterologous expression in yeast. Arabidopsis mutants in CYP86A8 [42] and CYP86A2 [43] are characterized by phenotypes involving defective cuticle formation, but the suberin of the mutants was not analyzed. Hence, it is still a matter of debate whether any of these v-hydroxylases are involved in suberin biosynthesis or not. The formation of a,v-diacids from v-OH-acids was demonstrated in suberizing tissue almost 30 years ago [44]. This dehydrogenase activity, which has been characterized and partially purified from suberizing potato tuber discs, was shown to be a two-step mechanism involving a v-oxo acid as intermediate (reviewed in [1]). The first enzyme might be encoded by HOTHEAD-like oxidoreductase genes, as indicated by the reduced amounts of a,v-diacids in the cutin of the hothead mutant [45]. However, the Current Opinion in Plant Biology 2007, 10:252–259
Our current knowledge of how these monomers are linked together, transferred to the apoplast and incorporated into the suberin polyester is insufficient. Glycerol- and ferulic-acid-dependent acyltransferases are required to form the oligomeric building blocks described above. In Arabidopsis, there are at least 19 potential acyltransferases that have been identified through sequence similarity [48]. Traditionally, building units for cell wall polymers were thought to be exported by a vesicular pathway from the endoplasmic reticulum to their extracellular destination. Recently, CER5, encoding an ABCtransporter, was identified as a factor in the transfer of aliphatic wax precursor across the plasma membrane [49]. Whether a direct export of aliphatic suberin precursor also occurs via an ABC-transporter, as hypothesized for longchain aliphatic wax precursors, remains to be elucidated. As deduced from cuticular defects in a mutant affected in the extracellular protein BODYGUARD, the formation of the suberin polymer by esterification probably involves members of the a,b-hydrolase superfamily, which contains hydrolases, esterases, cutinases and lipases [50]. Finally, the aliphatic polyester is linked to the polyaromatic domain via ferulic acid, presumably in a step catalyzed by cell wall peroxidases [51,52]. Suberin deposition occurs only in specific tissues and, in the case of Casparian bands, even at a specific site in the radial cell wall of a single cell. This and the induction of suberization in response to environmental conditions indicate a complex regulation of suberin biosynthesis, for which the molecular mechanisms are totally unknown to date. www.sciencedirect.com
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parsnip, rutabaga, turnip, red beet and sweet potato by combined gas-liquid chromatography and mass spectrometry. Plant Physiol 1975, 55:567-573.
Conclusions There is still a great gap in our knowledge of suberin biosynthesis. The core reactions in suberin biosynthesis, catalyzed by P450 and FAE complexes, take place at the endoplasmic reticulum membranes. The membraneassociation in complexes and the lack of commercially available substrates (e.g. long chain v-OH-acid-CoA) handicaps the progress of biochemical studies favoring molecular genetic approaches. The hypothetical pathway of suberin biosynthesis (Figure 1), which is based on knowledge of the chemical composition of Arabidopsis suberin [15] combined with a list of candidate genes for lipid metabolism [48], will lead to the identification of suberin mutants in future forward and reverse genetic approaches. This process will be supported by information available from tissue-specific root expression of candidate genes from genome [53] and family-focused (P450 [54]) microarray studies. The identification of the molecular elements of suberin formation might enable suberin engineering strategies to improve resistance to environmental stress conditions in agriculturally important plants. The unique properties of cork combine low permeability, low temperature conductivity, and high chemical resistance with high elasticity and low weight. These properties and cork’s chemical composition, which is dominated by long-chain aliphates with terminal functional groups, have drawn attention to the use of suberin biosynthetic products in industrial applications. These include the use of cork products in composite materials as sealant or insulators and the utility of plant-produced suberin monomers as bio-based lubricants or polyester foams [55].
References and recommended reading Papers of particular interest, published within the period of review, have been highlighted as: of special interest of outstanding interest 1.
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