Glycerol-derived ester oligomers from cork suberin

The cork suberin polyester was partially depolymerized by a methanolysis reaction catalyzed by calcium hydroxide. The methanolisate was analysed by ESI-MS/MS in the form of [M+Li](+) adduct-ions. This reaction solubilized a mixture of monomers and oligomers, including a set of glycerol-derived dimeric and trimeric esters. Four types of glycerol esters were identified: monoacylglycerols of alpha,omega-diacids, of omega-hydroxyacids and of monoacids; diglycerol diesters of alpha,omega-diacids; diacylglycerols of alpha,omega-diacids; monoacylglycerols of linear dimeric esters of alpha,omega-diacids and omega-hydroxyacids. The alpha,omega-diacids and omega-hydroxyacids found as monomer residues in the glycerol esters are the main ones found as cork suberin monomers. It is concluded that suberin is a glycerol-derived lipid of polymeric dimensions. Due to the protective and insulating role that it plays in plants, suberin should be considered together with the other known glycerolipids that build up biological membranes.     

Glycerol and glyceryl esters of omega-hydroxyacids in cutins

Cutins from the leaves and fruits of seven plant species were depolymerized by NaOCH(3)-methanolysis. The monomers that were released mostly included C16 and C18 omega-hydroxyacids with mid-chain oxygenated substitutions, namely epoxy and hydroxyl groups. Glycerol was also solubilized as a monomer in quantities that ranged from 1 to 14% of the methanolysates. Partial depolymerization of three cutins by CaO-methanolysis released the same monomers as had been obtained in the previous reaction, as well as small quantities of 1- and 2-monoacylglyceryl esters of omega-hydroxyacids. Molar proportions of glycerol permit the esterification of a significant part of the aliphatic omega-hydroxyacids, thereby possibly playing a major role in the polyester structure of cutin. Glycerol had not previously been known to form part of the cutin polymer.     

Apoplastic polyesters in Arabidopsis surface tissues--a typical suberin and a particular cutin

Cutinized and suberized cell walls form physiological important plant-environment interfaces as they act as barriers limiting water and nutrient loss and protect from radiation and invasion by pathogens. Due to the lack of protocols for the isolation and analysis of cutin and suberin in Arabidopsis, the model plant for molecular biology, mutants and transgenic plants with a defined altered cutin or suberin composition are unavailable, causing that structure and function of these apoplastic barriers are still poorly understood. Transmission electron microscopy (TEM) revealed that Arabidopsis leaf cuticle thickness ranges from only 22 nm in leaf blades to 45 nm on petioles, causing the difficulty in cuticular membrane isolation. We report the use of polysaccharide hydrolases to isolate Arabidopsis cuticular membranes, suitable for depolymerization and subsequent compositional analysis. Although cutin characteristic omega-hydroxy acids (7%) and mid-chain hydroxylated fatty acids (8%) were detected, the discovery of alpha,omega-diacids (40%) and 2-hydroxy acids (14%) as major depolymerization products reveals a so far novel monomer composition in Arabidopsis cutin, but with chemical analogy to root suberin. Histochemical and TEM analysis revealed that suberin depositions were localized to the cell walls in the endodermis of primary roots and the periderm of mature roots of Arabidopsis. Enzyme digested and solvent extracted root cell walls when subjected to suberin depolymerization conditions released omega-hydroxy acids (43%) and alpha,omega-diacids (24%) as major components together with carboxylic acids (9%), alcohols (6%) and 2-hydroxyacids (0.1%). This similarity to suberin of other species indicates that Arabidopsis roots can serve as a model for suberized tissue in general.     

Changing the Dimensions of Suberin Lamellae of Green Cotton Fibers with a Specific Inhibitor of the Endoplasmic Reticulum-Associated Fatty Acid Elongases

The fibers of the green lint mutant of cotton (Gossypium hirsutum L.) contain large amounts of wax and are suberized. More than 96% of the bifunctional aliphatic suberin monomers ([alpha],[omega]-alkanedioic acids and [omega]-hydroxyalkanoic acids) have chain lengths of C22 and C24 in green cotton fiber suberin. In fibers grown in the presence of S-ethyl-N,N-dipropylthiocarbamate (EPTC), a specific inhibitor of the endoplasmic reticulum-associated fatty acid elongases, the aliphatic suberin monomers were shortened to chain lengths of C16 and C18. Whereas the amounts of most suberin monomers were not negatively affected by the inhibitor treatment, the amounts of [alpha],[omega]-alkanedioic acids and of glycerol were reduced by more than 80%. Analysis in the transmission electron microscope showed a reduction in suberin content after EPTC treatment. The suberin layers were discontinuous and consisted of fewer lamellae than in the controls. A small proportion (up to 22%) of the electron-translucent suberin lamellae were thinner after EPTC treatment, probably because of the shortening of the aliphatic suberin monomers. A larger proportion of the electron-translucent lamellae were thicker than the lamellae in the controls. Possible explanations for this observation are discussed.     

Effects of NO 3 deficiency and NaCl stress on suberin deposition in rhizo- and hypodermal (RHCW) and endodermal cell walls (ECW) of castor bean (Ricinus communis L.) roots

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.     

Soybean root suberin: anatomical distribution, chemical composition, and relationship to partial resistance to Phytophthora sojae

Soybean (Glycine max L. Merr.) is a versatile and important agronomic crop grown worldwide. Each year millions of dollars of potential yield revenues are lost due to a root rot disease caused by the oomycete Phytophthora sojae (Kaufmann & Gerdemann). Since the root is the primary site of infection by this organism, we undertook an examination of the physicochemical barriers in soybean root, namely, the suberized walls of the epidermis and endodermis, to establish whether or not preformed suberin (i.e. naturally present in noninfected plants) could have a role in partial resistance to P. sojae. Herein we describe the anatomical distribution and chemical composition of soybean root suberin as well as its relationship to partial resistance to P. sojae. Soybean roots contain a state I endodermis (Casparian bands only) within the first 80 mm of the root tip, and a state II endodermis (Casparian bands and some cells with suberin lamellae) in more proximal regions. A state III endodermis (with thick, cellulosic, tertiary walls) was not present within the 200-mm-long roots examined. An exodermis was also absent, but some walls of the epidermal and neighboring cortical cells were suberized. Chemically, soybean root suberin resembles a typical suberin, and consists of waxes, fatty acids, omega-hydroxy acids, alpha,omega-diacids, primary alcohols, and guaiacyl- and syringyl-substituted phenolics. Total suberin analysis of isolated soybean epidermis/outer cortex and endodermis tissues demonstrated (1) significantly higher amounts in the endodermis compared to the epidermis/outer cortex, (2) increased amounts in the endodermis as the root matured from state I to state II, (3) increased amounts in the epidermis/outer cortex along the axis of the root, and (4) significantly higher amounts in tissues isolated from a cultivar ('Conrad') with a high degree of partial resistance to P. sojae compared with a susceptible line (OX760-6). This latter correlation was extended by an analysis of nine independent and 32 recombinant inbred lines (derived from a 'Conrad' x OX760-6 cross) ranging in partial resistance to P. sojae: Strong negative correlations (-0.89 and -0.72, respectively) were observed between the amount of the aliphatic component of root suberin and plant mortality in P. sojae-infested fields.     

Hydroxycinnamates in suberin formation

Hydroxycinnamates are found associated with suberin in several forms: covalently linked to the aliphatic suberin; in the residue after suberin-removal; and in the non-polar extractives of suberized tissues in the form of alkyl ferulates. Suberin-associated hydroxycinnamates have been found mainly as ferulic acid-derivatives, sometimes as feruloylamides and in a lesser extent as caffeates. Ferulic acid esters of long-chain ω-hydroxyacids are prevalent in the partial depolymerisation products of suberin. Also, enzymes able to catalyze the feruloylation of ω-hydroxyacids were found timely-associated with the suberization process. It is proposed that ferulic acid, and its dimers, through esterification to ω-hydroxyacids, covalently link the suberin aliphatic polyester to suberin-associated polyaromatics. In this case, the known role of ferulates, and related hydroxycinnamates, as cross-linkers of structurally different polymers would be enlarged to suberized cell-walls.     

The lipid polyester composition of Arabidopsis thaliana and Brassica napus seeds

Mature seeds of Arabidopsis thaliana and Brassica napus contain a complex mixture of aliphatic monomers derived from the non-extractable lipid polyesters deposited by various seed tissues. Methods of polyester depolymerization of solvent-extracted seeds and analysis of aliphatic monomers were compared. Sodium methoxide-catalyzed depolymerization, followed by GC analysis of the acetylated monomers, was developed for routine quantitative analysis suitable for 0.5g seed samples. In Arabidopsis seeds, the major C16 and C18 monomers identified included omega-hydroxy fatty acids and alpha,omega-dicarboxylic acids derived from palmitate, oleate and linoleate, and 9,10,18-trihydroxyoctadecenoic acid. Among monomers which can collectively be considered likely to be derived from suberin, docosan-1-ol, docosane-1,22-diol, 22-hydroxydocosanoic acid, 24-hydroxytetracosanoic acid, tetracosane-1,24-dioic acid and ferulic acid were the major species. Compared to Arabidopsis, Brassica seeds showed a roughly similar proportion of monomer classes, with the exception that alkan-1ols were 3-fold higher. Also, there were much less C24 aliphatic species and significant amounts of C14-C16 alkan-1ols, including iso- and anteiso-methyl branched compounds. Dissection and analysis of mature Brassica seeds showed that the trihydroxy C18:1 fatty acid was found mainly in the embryo, while ferulate, fatty alcohols and C22 and C24 species were specific to the seed coat plus endosperm.     

The Arabidopsis cytochrome P450 CYP86A1 encodes a fatty acid omega-hydroxylase involved in suberin monomer biosynthesis

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 length 相似文献   

Epoxyoctadecanoic acids in plant cutins and suberins

Three C₁₈ epoxy acids occur in plant cutins and suberins. 9,10-Epoxy-18-hydroxyoctadecanoic acid is a common constituent of both cutins and suberins whilst 9,10-epoxy-18-hydroxyoctadec-12-enoic acid is also present in some cutins. 9,10-Epoxyoctadecane-1,18-dioic acid occurs more commonly in suberins. Epoxy acids may comprise up to 60% of the total monomers obtained from some polymers. The epoxy compounds are readily converted into their corresponding alkoxyhydrin alkyl esters on depolymerization of cutin or suberin by alcoholysis. The chromatographic and MS properties of the alkoxyhydrin derivatives enable them to be readily distinguished from other cutin and suberin hydroxyfatty acids and to be used for the qualitative and quantitative determination of epoxy acids in the polymers.     

The chemical composition of suberin in apoplastic barriers affects radial hydraulic conductivity differently in the roots of rice (Oryza sativa L. cv. IR64) and corn (Zea mays L. cv. Helix)

Apoplastic transport barriers in the roots of rice (Oryza sativa L. cv. IR64) and corn (Zea mays L. cv. Helix) were isolated enzymatically. Following chemical degradation (monomerization, derivatization), the amounts of aliphatic and aromatic suberin monomers were analysed quantitatively by gas chromatography and mass spectrometry. In corn, suberin was determined for isolated endodermal (ECW) and rhizo-hypodermal (RHCW) cell walls. In rice, the strong lignification of the central cylinder (CC), did not allow the isolation of endodermal cell walls. Similarly, exodermal walls could not be separated from the rhizodermal and sclerenchyma cell layers. Suberin analyses of ECW and RHCW of rice, thus, refer to either the entire CC or to the entire outer part of the root (OPR), the latter lacking the inner cortical cell layer. In both species, aromatic suberin was mainly composed of coumaric and ferulic acids. Aliphatic suberin monomers released from rice and corn belonged to five substance classes: primary fatty acids, primary alcohols, diacids, omega-hydroxy fatty acids, and 2-hydroxy fatty acids, with omega-hydroxy fatty acids being the most prominent substance class. Qualitative composition of aliphatic suberin of rice was different from that of corn; (i) it was much less diverse, and (ii) besides monomers with chain lengths of C(16), a second maximum of C(28) was evident. In corn, C(24) monomers represented the most prominent class of chain lengths. When suberin quantities were related to surface areas of the respective tissues of interest (hypodermis and/or exodermis and endodermis), exodermal cell walls of rice contained, on average, six-times more aliphatic suberin than those of corn. In endodermal cell walls, amounts were 34 times greater in rice than in corn. Significantly higher amounts of suberin detected in the apoplastic barriers of rice corresponded with a substantially lower root hydraulic conductivity (Lp(r)) compared with corn, when water flow was driven by hydrostatic pressure gradients across the apoplast. As the OPR of rice is highly porous and permeable to water, it is argued that this holds true only for the endodermis. The results imply that some caution is required when discussing the role of suberin in terms of an efficient transport barrier for water. The simple view that only the quantity of suberin present is important, may not hold. A more detailed consideration of both the chemical nature of suberins and of the microstructure of deposits is required, i.e. how suberins impregnate wall pores.     

Glycerol is a suberin monomer. New experimental evidence for an old hypothesis

The monomer composition of the esterified part of suberin can be determined using gas chromatography-mass spectroscopy technology and is accordingly believed to be well known. However, evidence was presented recently indicating that the suberin of green cotton (Gossypium hirsutum cv Green Lint) fibers contains substantial amounts of esterified glycerol. This observation is confirmed in the present report by a sodium dodecyl sulfate extraction of membrane lipids and by a developmental study, demonstrating the correlated accumulation of glycerol and established suberin monomers. Corresponding amounts of glycerol also occur in the suberin of the periderm of cotton stems and potato (Solanum tuberosum) tubers. A periderm preparation of wound-healing potato tuber storage parenchyma was further purified by different treatments. As the purification proceeded, the concentration of glycerol increased at about the same rate as that of α,ω-alkanedioic acids, the most diagnostic suberin monomers. Therefore, it is proposed that glycerol is a monomer of suberins in general and can cross-link aliphatic and aromatic suberin domains, corresponding to the electron-translucent and electron-opaque suberin lamellae, respectively. This proposal is consistent with the reported dimensions of the electron-translucent suberin lamellae.     

Metabolism of cholestane-3 beta,5 alpha,6 beta-triol. II. Identification of two major neutral metabolites in the rat

Rats were given a single oral dose of cholestane-3beta,5alpha,6beta-triol-4-(14)C, and their feces were collected. The two major neutral metabolites were separated and isolated by use of solvent fractionation and chromatographic methods. The metabolites were identified as cholestane-3beta,5alpha-diol-6-one and a mixture of long-chain fatty acid esters of cholestane-3beta,5alpha,-6beta-triol. Cholestane-3beta,5alpha-diol-6-one was identified using thin-layer and gas-liquid chromatography, infrared spectroscopy, and the spectrum produced by reaction with 65% sulfuric acid. The mixed esters of cholestane-3beta,5alpha,6beta-triol were subjected to basic hydrolysis, and the steroid moiety was identified using the same techniques employed for cholestane-3beta,5alpha-diol-6-one. The fatty acids were analyzed by gas-liquid chromatography of their methyl esters.     

Folding of a de novo designed native-like four-helix bundle protein

The folding of a model native-like dimeric four-helix bundle protein, (alpha(2))(2), was investigated using guanidine hydrochloride, hydrostatic pressure, and low temperature. Unfolding by guanidine hydrochloride followed by circular dichroism and intrinsic fluorescence spectroscopy revealed a highly cooperative transition between the native-like and unfolded states, with free energy of unfolding determined from CD data, DeltaG(unf) = 14.3 +/- 0.8 kcal/mol. However, CD and intrinsic fluorescence data were not superimposable, indicating the presence of an intermediate state during the folding transition. To stabilize the folding intermediate, we used hydrostatic pressure and low temperature. In both cases, dissociation of the dimeric native-like (alpha(2))(2) into folded monomers (alpha(2)) was observed. van't Hoff analysis of the low temperature experiments, assuming a two-state dimer 171-monomer transition, yielded a free energy of dissociation of (alpha(2))(2) of DeltaG(diss) = 11.4 +/- 0.4 kcal/mol, in good agreement with the free energy determined from pressure dissociation experiments (DeltaG(diss) = 10.5 +/- 0.1 kcal/mol). Binding of the hydrophobic fluorescent probe 4,4'-dianilino-1,1'-binaphthyl-5,5'-disulfonic acid (bis-ANS) to the pressure- and cold-dissociated states of (alpha(2))(2) indicated the existence of molten-globule monomers. In conclusion, we demonstrate that the folding pathway of (alpha(2))(2) can be described by a three-state transition including a monomeric molten globule-like state.     

Glomerular basement membrane. Identification of dimeric subunits of the noncollagenous domain (hexamer) of collagen IV and the Goodpasture antigen

The noncollagenous (NC1) domain hexamer of glomerular basement membrane (GBM) collagen is composed of a multiplicity of monomeric and dimeric subunits, and specific subunits are the targets for anti-GBM autoantibodies of patients with Goodpasture (GP) syndrome. The identity of GBM monomers has been established and the alpha 3(IV)NC1 monomer identified as the one that binds GP antibodies (Gunwar, S., Saus, J., Noelken, M. E., and Hudson, B. G. (1990) J. Biol. Chem. 265, 5466-5469). In the present study, the chain origin of 25 dimeric components and the identity of those that bound the anti-GBM antibodies from two GP patients were determined. This was accomplished by NH2-terminal sequence analysis and immunoblotting analysis of dimeric components that were resolved by two-dimensional electrophoresis in combination with high pressure liquid chromatography. The results revealed that (a) the components are mainly homodimers of the NC1 domains of alpha 1, alpha 2, alpha 3, alpha 4, and probably alpha 5 chains of collagen IV, reflecting a specificity of promoter-promoter association and (b) each homodimer had several size and charge isoforms. The GP antibodies bound exclusively to both alpha 3(IV)NC1 monomers and dimers and not to other basement membrane constituents. These findings provided new insights about the structure of GBM collagen and together with our previous findings firmly established the alpha 3(IV) chain as the target for the anti-GBM antibodies that mediate glomerulonephritis and pulmonary hemorrhage in patients with Goodpasture syndrome.     

Water and solute permeabilities of Arabidopsis roots in relation to the amount and composition of aliphatic suberin

Although it is implied that suberized apoplastic barriers of roots negatively correlate with water and solute permeabilities, direct transport measurements across roots with altered amounts and compositions of aliphatic suberin are scarce. In the present study, hydroponically grown Arabidopsis wild types (Col8 and Col0) and different suberin mutants with altered amounts and/or compositions (horst, esb1-1, and esb1-2) were used to test this hypothesis. Detailed histochemical studies revealed late development of Casparian bands and suberin lamellae in the horst mutant compared with wild types and esb mutants. Suberin analysis with gas chromatography and mass spectrometry (GC-MS) showed that the horst mutant had ～33% lower amounts of aliphatic monomers than Col8 and Col0. In contrast, enhanced suberin mutants (esb1-1 and esb1-2) had twice the amount of suberin as the wild types. Correlative permeability measurements, which were carried out for the first time with a root pressure probe for Arabidopsis, revealed that the hydraulic conductivity (Lp(r)) and NaCl permeability (P(sr)) of the whole root system of the horst mutant were markedly greater than in the respective wild types. This was reflected by the total amounts of aliphatic suberin determined in the roots. However, increased levels of aliphatic suberin in esb mutants failed to reduce either water or NaCl permeabilities below those of the wild types. It was concluded that the simple view and the conventional assumption that the amount of root suberin negatively correlates with permeability may not always be true. The aliphatic monomer arrangement in the suberin biopolymer and its microstructure also play a role in apoplastic barrier formation.     

An untargeted liquid chromatography–mass spectrometry‐based workflow for the structural characterization of plant polyesters

Cell wall localized heterogeneous polyesters are widespread in land plants. The composition of these polyesters, such as cutin, suberin, or more plant‐specific forms such as the flax seed coat lignan macromolecule, can be determined after total hydrolysis of the ester linkages. The main bottleneck in the structural characterization of these macromolecules, however, resides in the determination of the higher order monomer sequences. Partial hydrolysates of the polyesters release a complex mixture of fragments of different lengths, each present in low abundance and therefore are challenging to structurally characterize. Here, a method is presented by which liquid chromatography–mass spectrometry (LC‐MS) profiles of such partial hydrolysates are searched for pairs of related fragments. LC‐MS peaks that show a mass difference corresponding to the addition of one or more macromolecule monomers were connected in a network. Starting from the lowest molecular weight peaks in the network, the annotation of the connections as the addition of one or more polyester monomers allows the prediction of consecutive and increasingly complex adjacent peaks. Multi‐stage MS (MSn) experiments further helped to reject, corroborate, and sometimes refine the structures predicted by the network. As a proof of concept, this procedure was applied to partial hydrolysates of the flax seed coat lignan macromolecule, and allowed to characterize 120 distinct oligo‐esters, consisting of up to six monomers, and containing monomers and linkages for which incorporation in the lignan macromolecule had not been described before. These results showed the capacity of the approach to advance the structural elucidation of complex plant polyesters.     

A feruloyl transferase involved in the biosynthesis of suberin and suberin‐associated wax is required for maturation and sealing properties of potato periderm

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 ω‐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 ω‐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.