Unraveling ferulate role in suberin and periderm biology by reverse genetics

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.Key words: BAHD feruloyl acyltransferases, ferulate, periderm, potato tuber skin, suberin, suberized tissues, waxRecently published reverse genetic studies in Arabidopsis and potato identified two orthologous genes that encode a BAHD feruloyl transferase acting on aliphatics and showed that deficiency in these enzymes produces a decrease in suberin-associated ferulic acid. These results, here discussed, signify an important advance in suberin biochemistry and ultrastructure, providing a valuable new insight into the organization of the suberized tissues and their role in the control of water vapour loss.     

Following Suberization in Potato Wound Periderm by Histochemical and Solid-State 13C Nuclear Magnetic Resonance Methods

The time course of suberization in wound periderm from potato (Solanum tuberosum L.) has been monitored by histochemical and high-resolution solid-state nuclear magnetic resonance (NMR) methods. Light microscopy conducted after selective staining of the lipid and double-bonded constituents shows that suberin is deposited at the outermost intact cell-wall surface during the first 7 d of wound healing; suberization forms a barrier to tissue infiltration at later times. Cross polarization-magic angle spinning 13C NMR spectra demonstrate the deposition of a polyester containing all major suberin functional groups after just 4 d of wound healing. Initially the suberin includes a large proportion of aromatic groups and fairly short aliphatic chains, but the spectral data demonstrate the growing dominance of long-chain species during the period 7 to 14 d after wounding. The results of preliminary 13C-labeling experiments with sodium [2-13C]acetate and DL-[1-13C]phenylalanine provide an excellent prospectus for future NMR-based studies of suberin biosynthesis.     

The ABC Transporter ABCG1 Is Required for Suberin Formation in Potato Tuber Periderm

The lipid biopolymer suberin plays a major role as a barrier both at plant-environment interfaces and in internal tissues, restricting water and nutrient transport. In potato (Solanum tuberosum), tuber integrity is dependent on suberized periderm. Using microarray analyses, we identified ABCG1, encoding an ABC transporter, as a gene responsive to the pathogen-associated molecular pattern Pep-13. Further analyses revealed that ABCG1 is expressed in roots and tuber periderm, as well as in wounded leaves. Transgenic ABCG1-RNAi potato plants with downregulated expression of ABCG1 display major alterations in both root and tuber morphology, whereas the aerial part of the ABCG1-RNAi plants appear normal. The tuber periderm and root exodermis show reduced suberin staining and disorganized cell layers. Metabolite analyses revealed reduction of esterified suberin components and hyperaccumulation of putative suberin precursors in the tuber periderm of RNA interference plants, suggesting that ABCG1 is required for the export of suberin components.     

Response of xylem parenchyma by suberization in some hardwoods after mechanical injury

Summary Wound responses of xylem parenchyma by suberization were investigated in some hardwoods by light and electron microscopy. Suberized ray and axial parenchyma cells form a distinct boundary around the wound in all investigated species. Vessels and fibres within and close behind the suberized area appeared more or less occluded; vessels in Fagus, Quercus, and Populus contained suberized tyloses, those in Betula and Tilia contained amorphous and fibrillar deposits. A common mechanism for suberin deposition in the parenchyma cells became evident. Cisternae of the endoplasmic reticulum were apparently involved in suberization. Suberin compounds are extruded by cytoplasmic vesicles, which fused with the plasma membrane, in order to release their content. The suberin layer exhibited the typical lamellated structure; cytoplasmic continuity between suberized cells by plasmodesmata was maintained through the suberin layer. Fagus revealed the most intense suberized area as compared with the other species. Within the reaction zone of Fagus and Quercus, some individual ray and axial parenchyma cells exhibited a subdivision into 2 or 3 compartments prior to suberization. Subdivision was achieved by the formation of a primary wall-like layer. Subsequently, the compartments became individually suberized. Wounding during winter did not induce suberization. Also, samples wounded and kept under water during the vegetation period showed no response. The role of suberization in the effectivity of wound-associated compartmentalization is discussed.     

Magnesium deficiency results in increased suberization in endodermis and hypodermis of corn roots

The composition of the aliphatic components of suberin in the stele and cortex of young corn (Zea mays L.) roots was determined by combined gas-liquid chromatography/mass spectrometry of the LiAlD₄ depolymerization products. ω-Hydroxy acids were shown to be the major class of the aliphatic components of both the hypodermal (35%) and endodermal (28%) polymeric materials with the dominant chain length being C₂₄ in the former and C₁₆ in the latter. Nitrobenzene oxidation of the roots generated p-hydroxybenzaldehyde and vanillin with much less syringaldehyde. Electron microscopic examination of the hypodermal and endodermal cell walls from roots of corn plants grown in a Mg²⁺ -deficient (0.03 millimolar) nutrient solution showed that these walls were more heavily suberized than the analogous walls of roots from plants grown in normal (2 millimolar) Mg²⁺ levels. Analysis of the LiAlD₄ depolymerization products of the suberin polymers from these roots showed that the roots grown in low Mg²⁺ had 3.5 times as much aliphatic suberin monomers on a weight basis as the roots from plants grown in nutrient with normal Mg²⁺ levels. Roots from plants grown in Mg²⁺ -deficient nutrient solution released 3.8 times the amount of aromatic aldehydes upon nitrobenzene oxidation as that released from normal roots. As the degree of Mg²⁺ deficiency of the nutrient solution was increased, there was an increase in the aliphatic and aromatic components characteristic of suberin. Thus, both ultrastructural and chemical evidence strongly suggested that Mg²⁺ deficiency resulted in increased suberization of the cell walls of both hypodermis and endodermis of Zea mays roots. The roots from Mg²⁺ -deficient plants also had a higher amount of peroxidase activity when compared to control roots.     

Fine structure of isolated and non-isolated potato tuber periderm

Cell walls of the periderm of native potato tuber (Solanum tuberosum L. cv. Primura) consist of a primary wall, a suberized secondary wall and a tertiary wall. With a mixture of pectinase and cellulase intact periderm membranes can be isolated. Isolation does not affect fine structure. It is suggested that the lignin in the middle lamellae and primary walls prevents the enzymes from digesting pectinaceous materials and cellulose. In specimens fixed with OsO₄, the suberized walls appear as alternating electrondense and electron-lucent lamellae. This lamellar architecture is not altered by extraction with chloroform. Therefore, the current view that the electronlucent lamellae consist of soluble lipids (waxes) can no longer be maintained. It is argued that the lamellation is a property of the suberin itself, and the suberized wall consists of alternating layers of suberins differing in polarity. A hypothesis of suberin assembly from sub-units is advanced and the subunits are shown for the first time.     

ULTRASTRUCTURE OF THE EPIDERMIS OF DEVELOPING COTTON (GOSSYPIUM) SEEDS: SUBERIN,PITS, PLASMODESMATA,AND THEIR IMPLICATION FOR ASSIMILATE TRANSPORT INTO COTTON FIBERS

The basal part of cotton fibers (Gossypium arboreum and G. hirsutum) was studied with light and electron microscopy in order to improve the understanding of assimilate transport into the fibers during the deposition of the cellulosic secondary wall. Although the distal parts of white cotton fibers are not suberized, a variable amount of suberin was found at the fiber base. This suberin is typically deposited in concentric layers, alternating with polysaccharides. Numerous pits occur in the base of cotton fibers and in ordinary epidermal cells in the periclinal and anticlinal walls. About 25% of the length of periclinal walls is occupied by pits, but only 2% of the anticlinal walls, being pitted mainly in their proximal part. In suberized walls the deposition of suberin is not reduced in the pit region. The pits, whether or not suberized, contain plasmodesmata (22 ± 2.3 and 27 ± 3.3 · μm^−-2 in the periclinal and anticlinal walls of the white lint cultivar of G. hirsutum). Transport of assimilates into the fibers through the symplast is therefore possible. This transport may occur directly from mesophyll cells to fibers, or indirectly via ordinary epidermal cells. The minimum amount of assimilates transported into individual fibers during the phase of secondary wall deposition could be estimated (1.3 pg · fiber^−-1 · sec^−-1), as well as the corresponding symplastic flux of assimilates through the periclinal cell wall, neglecting a possible transport through the anticlinal walls (10^−-3 pg · μm^−-2 · sec^−-1). It is postulated that in the green lint genotype of G. hirsutum and in wild cotton species (not studied in this paper), the uptake of assimilates into the fibers occurs through the symplast, the apoplastic pathway being excluded by the suberization of the fibers during secondary wall formation. Although cultivated, white-linted cotton species may use the same pathway, loading of assimilates from the apoplast is theoretically also possible, and the relative contribution of both pathways has to be determined experimentally.     

Suberized tyloses in trees: An ultrastructural and cytochemical study

The nature of the wall layers observed in suberized tyloses was studied in Populus basalmifera L., Ulmus americana L. and Quercus rubra L. As the suberin layers were present only in tyloses that had completed their expansion, most of the results concern mature tyloses. The cyto- and immunocytochemical tests were conducted, respectively, with an exoglucanase having a binding affinity for β(1→4)-D-glucans, the subunits of cellulose, and with two monoclonal antibodies specific for un-esterified and esterified pectic molecules. In the three species, labelling for pectic compounds was intense over the external layer of tyloses but usually more dispersed or nearly absent over the layer corresponding to a primary wall that was, however, intensely labelled for β(1→4)-D-glucans. The outer wall layer, comparable to a middle lamella in mature tyloses, was continuous with similar material that appeared to be secreted by the tylosis. This material was particularly abundant in pit chambers, in void spaces between the tylosis and the vessel wall, particularly at the junction of the vessel and two adjacent cells, and close to the rim of vessel perforation plates. In P. balsamifera, a single suberized layer or occasionally a succession of suberized and cellulose-containing layers was observed internal to the tylosis primary wall. In U. americana, the wall of tylosis was similar to that of P. balsamifera except that, at times, a secondary-wall-like layer was formed and only a single suberized layer was observed. In Q. rubra, the suberized layer was always observed internal to the tylosis secondary wall. Simple pits were also constantly noted in Q. rubra tyloses. The occasional occurrence of a cellulosic layer internally to the suberized layer was observed in the three species. Histochemical tests revealed that lignin was also an important component of the tylosis wall. The tyloses frequently contained phenolic compounds in close association with the suberized layers. The significance of the formation of suberized tyloses in trees is discussed.     

Chemical and ultrastructural evidence that waxes associated with the suberin polymer constitute the major diffusion barrier to water vapor in potato tuber (Solanum tuberosum L.)

Combined gas chromatography-mass spectrometry showed that C₂₁, C₂₃, and C₂₅ n-alkanes accumulated in the suberized layers during wound healing of cores of potato tuber tissue. Treatment (10 min) of freshly-cut tissue with trichloroacetate (TCA), an inhibitor of fatty-acid chain elongation, severely inhibited accumulation of hydrocarbons and fatty alcohols associated with the suberized layer in the wound healing tissue (maximum inhibition at 4 mM) but had very little effect on the deposition of the major aliphatic components of the suberin polymer. This preferential inhibition of wax synthesis resulted in severe inhibition of the development of diffusion resistance of the tissue to water vapor. These results strongly indicate that the waxes associated with the suberin polymer, rather than the polymer itself, consitute the major diffusion barrier formed during wound healing. Electron-microscopic examination showed that inhibition of wax synthesis by TCA disrupted the formation of the lamellar structure of suberin specifically by preventing the formation of the light bands. This evidence strongly suggests that the light bands in the suberin complex are composed of waxes.Scientific Paper No. 5330, Project 2001, College of Agriculture Research Center, Washington State University, Pullman, Washington 99164, USA     

Spectral Snapshots of Bacterial Cell-Wall Composition and the Influence of Antibiotics by Whole-Cell NMR

Gram-positive bacteria surround themselves with a thick cell wall that is essential to cell survival and is a major target of antibiotics. Quantifying alterations in cell-wall composition are crucial to evaluating drug modes of action, particularly important for human pathogens that are now resistant to multiple antibiotics such as Staphylococcus aureus. Macromolecular and whole-cell NMR spectroscopy allowed us to observe the full panel of carbon and nitrogen pools in S. aureus cell walls and intact whole cells. We discovered that one-dimensional ¹³C and ¹⁵N NMR spectra, together with spectroscopic selections based on dipolar couplings as well as two-dimensional spin-diffusion measurements, revealed the dramatic compositional differences between intact cells and cell walls and allowed the identification of cell-wall signatures in whole-cell samples. Furthermore, the whole-cell NMR approach exhibited the sensitivity to detect distinct compositional changes due to treatment with the antibiotics fosfomycin (a cell-wall biosynthesis inhibitor) and chloramphenicol (a protein synthesis inhibitor). Whole cells treated with fosfomycin exhibited decreased peptidoglycan contributions while those treated with chloramphenicol contained a higher percentage of peptidoglycan as cytoplasmic protein content was reduced. Thus, general antibiotic modes of action can be identified by profiling the total carbon pools in intact whole cells.     

Phellogen Regeneration in Injured Peach Tree Bark

Injury to peach bark phellogen leads to the generation of newtissues and the re-establishment of meristematic continuity.Two types of tissue changes after wounding were identified andquantified in bark of seven peach clones: (1) cell wall modifications(lignification and suberization) of tissues present at the timeof wounding, and (2) generation of the new phellogen and itsderivatives. Tissue responses were quantified with a microscopephotometer using selective histochemistry and autofluorescenceto detect lignin and suberin deposition over time. Suberin continuitywas re-established via suberin deposition in a layer of cells,present at the time of wounding, approximately 800 µminternal to the wound surface. Phellogen continuity was re-establishedimmediately internal to and abutting the suberized tissue. Thenew phellogen gave rise to suberized phellem which, in its outwardexpansion, crushed the suberized boundary zone tissue formedearlier. All injured peach clones produced the same sequenceof tissue changes, although timing and degree of response variedwith clone and time of year. Differentiation, impervious tissue, lignin, Prunus persica (L.) Batsch, suberin, wounding     

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.     

Oritavancin exhibits dual mode of action to inhibit cell-wall biosynthesis in Staphylococcus aureus

Solid-state NMR measurements performed on intact whole cells of Staphylococcus aureus labeled selectively in vivo have established that des-N-methylleucyl oritavancin (which has antimicrobial activity) binds to the cell-wall peptidoglycan, even though removal of the terminal N-methylleucyl residue destroys the d-Ala-d-Ala binding pocket. By contrast, the des-N-methylleucyl form of vancomycin (which has no antimicrobial activity) does not bind to the cell wall. Solid-state NMR has also determined that oritavancin and vancomycin are comparable inhibitors of transglycosylation, but that oritavancin is a more potent inhibitor of transpeptidation. This combination of effects on cell-wall binding and biosynthesis is interpreted in terms of a recent proposal that oritavancin-like glycopeptides have two cell-wall binding sites: the well-known peptidoglycan d-Ala-d-Ala pentapeptide stem terminus and the pentaglycyl bridging segment. The resulting dual mode of action provides a structural framework for coordinated cell-wall assembly that accounts for the enhanced potency of oritavancin and oritavancin-like analogues against vancomycin-resistant organisms.     

Development of mestome sheath cells in leaves ofAegilops comosa var.thessalica

Summary The development of mestome sheath cells ofAegilops comosa var.thessalica was studied by electron microscopy. Anatomical and cytological observations show that this grass belongs to the C₃ or non-Kranz plants. In the asymmetrically thickened walls of mestome sheath cells a suberized lamella is present. This lamella is deposited asynchronously. In the midrib and the large lateral bundles it appears first in the outer and inner walls and usually later in the radial walls. In the small lateral bundles its appearance is delayed in the inner walls of those cells situated on the xylem side. At maturity the suberized lamella is observed in all cell walls; however, in the small lateral bundles it is partly or totally absent from the walls of some cells situated on the xylem side. Tertiary wall formation is asynchronous as well, for it generally follows the deposition pattern of the suberized lamella.During the development of the mestome sheath cells microtubules show marked changes in their number and orientation, being fewer and longitudinal during suberin deposition. Dictyosomes are very active and may be involved in primary and tertiary wall formation. Endoplasmic reticulum cisternae are abundant and partly smooth, while plasmalemmasomes may function to reduce the plasmalemma extension. However, cytoplasmic structures that are clearly involved in suberin synthesis could not be identified.Suberized lamellae react strongly with silver hexamine. This is probably due to post-fixation with osmium tetroxide.On the basis of structural characteristics the mestome sheath may be regarded as an endodermis (cf., alsoFahn 1974). The significance of this view for water and assimilate exchange between the mesophyll and the bundle is discussed.This report represents a portion of a doctoral dissertation.     

Permeability of the suberized mestome sheath in winter rye

Mestome sheath cells of winter rye (Secale cereale L. cv Puma) deposit suberized lamellae in their secondary cell walls. Histochemical tests including acid digestion and staining with Sudan IV and Chelidonium majus root extract were used to detect the presence of suberin in the primary cell wall. There was no evidence of a Casparian band between adjacent mestome sheath cells. Fluorescent dye techniques were used to trace solute movement through the rye leaf apoplast. Calcofluor white M2R, a fluorescent dye which binds to cell walls as it moves apoplastically, proved to be too limited in its mobility in leaves to test mestome sheath permeability. Trisodium 3-hydroxy-5,8,10 pyrene trisulfonate, a fluorescent dye which is mobile in the apoplast, moved easily up the vascular bundles in the transpiration stream, and diffused outward from the veins to the epidermal cell walls within minutes of reaching a particular level in the leaf. We conclude that the suberized mestome sheath of rye leaves is freely permeable to solutes moving apoplastically through radial primary cell walls.     

A suberized perimedullary reaction zone in Populus balsamifera novel for compartmentalization in trees

 Following artificial inoculation of nonhost Populus balsamifera with Ophiostoma ulmi, structural defensive tissues were formed in the xylem. Among these tissues there was a perimedullary sheath of cells, located adjacent to the invaded xylem, that originated from the dedifferentiation of perimedullary and xylem parenchyma cells. Histochemical tests revealed that this sheath was intensively suberized. A band of lignified cells was frequently detected on both sides of this suberized tissue. The formation of such a tissue at the pith margin represents a new type of anatomical barrier in relation to compartmentalization processes described for trees. Ultrastructural examination showed that the wall of cells forming this zone was generally composed of a compound middle lamella, a suberized secondary wall and a tertiary wall layer. Using colloidal gold conjugated to monoclonal antibodies against pectin and to an exoglucanase for cellulose, only limited labelling was obtained for pectin whereas labelling for cellulose was abundant in the compound middle lamella and the tertiary wall layer. In a few fibres close to this suberized zone, the latter probe also made it possible to distinguish the occasional presence of several alternating wall layers mainly composed of either suberin or cellulose. In Salix sp., another tree species belonging to the Salicaceae, this type of suberized reaction zone was also observed. The new reaction zone is similar in structure and location to a suberized barrier formed in nonwoody carnation (Dianthus caryophyllus) plants in the defense against vascular fungi. Received: 30 July 1996 / Accepted: 7 January 1997     

Abscisic Acid stimulation of suberization : induction of enzymes and deposition of polymeric components and associated waxes in tissue cultures of potato tuber

Effect of abscisic acid (ABA) on suberization of potato (Solanum tuberosum var. Russet-Burbank) tuber tissue culture was studied by measuring deposition of suberin components and the level of certain key enzymes postulated to be involved in suberization. ABA treatment resulted in a 3-fold increase in the polymeric aliphatic components of suberin and a 4-fold increase in the polymeric aromatic components. Hydrocarbons and fatty alcohols, two components characteristic of waxes associated with potato suberin, increased 9- and 5-fold, respectively, as a result of ABA treatment. Thus, the deposition of the polymeric aliphatics and aromatics as well as waxes, all of which have been postulated to be components of suberized cell walls, was markedly stimulated by ABA. ω-Hydroxy-fatty acid dehydrogenase which showed a rather high initial level of activity increased only 60% due to ABA treatment. Phenylalanine ammonia-lyase activity reached a maximum at a 5-fold level after 4 days in the ABA medium, whereas the control showed only a 3-fold increase. ABA treatment also resulted in a dramatic (7-fold) increase in an isozyme of peroxidase which has been specifically associated with suberization. Thus, ABA appears to induce certain key enzymes which are most probably involved in suberization.     

Bacterial cell wall composition and the influence of antibiotics by cell-wall and whole-cell NMR

The ability to characterize bacterial cell-wall composition and structure is crucial to understanding the function of the bacterial cell wall, determining drug modes of action and developing new-generation therapeutics. Solid-state NMR has emerged as a powerful tool to quantify chemical composition and to map cell-wall architecture in bacteria and plants, even in the context of unperturbed intact whole cells. In this review, we discuss solid-state NMR approaches to define peptidoglycan composition and to characterize the modes of action of old and new antibiotics, focusing on examples in Staphylococcus aureus. We provide perspectives regarding the selected NMR strategies as we describe the exciting and still-developing cell-wall and whole-cell NMR toolkit. We also discuss specific discoveries regarding the modes of action of vancomycin analogues, including oritavancin, and briefly address the reconsideration of the killing action of β-lactam antibiotics. In such chemical genetics approaches, there is still much to be learned from perturbations enacted by cell-wall assembly inhibitors, and solid-state NMR approaches are poised to address questions of cell-wall composition and assembly in S. aureus and other organisms.     

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

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