Wound-induced superoxide production and PAL activity decline with potato tuber age and wound healing ability

Wound healing of potato tubers involves the concerted action of several enzymes that facilitate polymerization of phenolics into suberin at the wound site. A decline in the efficiency of healing and resistance to pathogens with advancing tuber age was associated with reduced ability of older tubers to produce superoxide radicals (FRs) in response to wounding. Autophotographs of luminol‐treated longitudinal sections of tissue from 6‐, 18‐ and 30‐month‐old tubers revealed a substantial decline in superoxide production at the wound surface with advancing age. Older tubers were less able to respond to wounding by increasing phenylalanine ammonia lyase (PAL) activity. This enzyme produces t‐cinnamic acid, which constitutes a component of the phenolic domain of suberin, and is normally induced by wounding and/or ethylene. Interestingly, the ability of wounded tissue to oxidize exogenous 1‐aminocyclopropane‐1‐carboxylic acid (ACC) to C₂H₄ also decreased with advancing tuber age. The oxidation of ACC was inhibited by the FR scavenger, n‐propyl gallate (PG), and inhibition was greatest in tissue from younger tubers, reflecting their greater ability to produce superoxide radicals upon wounding. Regardless of tuber age, 1‐aminocyclobutane‐1‐carboxylic acid, an ACC oxidase inhibitor, did not inhibit C₂H₄ generation from exogenous ACC. Hence, C₂H₄ production from ACC by wounded tuber tissue is largely non‐enzymatic and FR‐driven, and thus serves as an indicator of the ability of wounded tissue to produce superoxide. Age‐induced reduction in PAL activity and FR production at the wound surface probably limited the oxidative polymerization of phenolics into suberin during wound periderm formation. The age‐induced loss in ability of wounded tissue to heal and resist pathogens is thus consistent with reduced synthesis and polymerization of phenolic adducts into suberin, a consequence of reduced FR and PAL activity at the wound surface.     

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.     

Composition,ultrastructure and function of the cutin- and suberin-containing layers in the leaf,fruit peel,juice-sac and inner seed coat of grapefruit (Citrus paradisi Macfed.)

Cutin and suberin polymers from various anatomical regions of grapefruit were analyzed chemically and ultrastructurally. The leaf, fruit peel and juice-sac showed an amorphous cuticular layer. The cutin in the leaf was composed of 10,16-dihydroxy C₁₆ acid and its positional isomers as the major monomers whereas 16-hydroxy-10-oxo C₁₆ acid was a major component in the fruit peel. Juice-sac cutin, on the other hand, contained the dihydroxy C₁₆ acids, hydroxyoxo C₁₆ acids, hydroxyepoxy C₁₈ acids and trihydroxy C₁₈ acids. Ultrastructural examination of the inner seed coat showed that an amorphous cuticular layer encircled the entire seed except in the chalazal region which showed several layers of cells with lamellar suberin structure throughout the cell walls. Consistent with the ultrastructural assignment, the compositions of the aliphatic components of the polymers from the chalazal region and the non-chalazal region indicated the presence of suberin and cutin, respectively. The aliphatic portion of the polymer from the chalazal region of the inner seed coat contained C₁₆, C_18:1, C₂₂ and C₂₄ -hydroxy acids (46% combined total) and the corresponding dicarboxylic acids (43%) as the major components. -Hydroxy-9,10-epoxy C₁₈ acids and 9,10,18-trihydroxy C₁₈ acids were the major components (77%) of the polymer from the non-chalazal portion of the inner seed coat. The main portion and the chalazal region of the inner seed coat yielded 17 and 342 g/cm² of aliphatic monomers, respectively, and the diffusion resistance of these two portions of the inner seed coat were 62 and 192 sec/cm, respectively. The inner seed coat was shown to be the major moisture diffusion barrier influencing imbibition and germination.Scientific Paper No. 5649, Project 2001, College of Agriculture Research Center, Washington State University, Pullman, Washington 99164     

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.     

Synthesis of Suberin during Wound-healing in Jade Leaves, Tomato Fruit, and Bean Pods

The structure and composition of the aliphatic monomers of the polymeric material deposited during wound-healing of tomato fruit, bean pods, and Jade leaves were examined. After removing the cuticle-containing layer of tissue, the wounds were healed for 14 days and the resulting surface layer was excised, lyophilized, solvent-extracted, and depolymerized by hydrogenolysis with LiAlH₄ or transesterified with BF₃ in methanol. The products obtained by the chemical depolymerization were subjected to thin layer chromatography and combined gas chromatography and mass spectrometry. The major aliphatic components isolated from the hydrogenolysate of the wound polymer produced by tomato fruit were hexadecane-1,16-diol and octadec-9-ene-1,18-diol, which were shown to be derived from a 1:1 mixture of ω-hydroxy and dicarboxylic acids of the appropriate chain length by LiAlH₄ reduction. Also identified in the wound polymer were long chain (>C₂₀) fatty acids and alcohols. This monomer composition is typical of suberin polymers and is in sharp contrast with that of the cutin of tomato fruit which contains dihydroxy C₁₆ acid as the major aliphatic component. The hydrogenolysis of the wound material from bean pods gave octadecene-1,18-diol as the major aliphatic component, and smaller amounts of hexadecane-1,16-diol and long chain alcohols. Similar treatment of the normal cuticular tissue of these pods gave hexadecane triol, as well as C₁₆ and C₁₈ alcohols. Hydrogenolysis of wound material from the Jade leaves gave octadecene-1,18-diol, C₁₆ and C₂₂ diols, as well as alcohols from C₁₆ to C₂₆, whereas similar treatment of the cutin-containing tissue from these leaves gave C₁₆ triol as the major aliphatic component. Thus, the major aliphatic monomers of the polymeric material deposited during the wound-healing of bean pods and Jade leaves are very similar to those of suberin, although the natural protective polymer of these tissues is cutin. From these results, it is concluded that suberization is a fundamental process involved in wound-healing in plants, irrespective of the chemical nature of the natural protective polymer of the tissue.     

Wax and suberin development of native and wound periderm of potato (<Emphasis Type="Italic">Solanum tuberosum</Emphasis> L.) and its relation to peridermal transpiration

Native and wound periderm was isolated enzymatically from potato (Solanum tuberosum L. cv. Desirée) tubers at different time intervals between 0 days up to 4 weeks after harvesting. Wound periderm formation was induced by carefully removing native periderm from freshly harvested tubers before storage. The chemical composition of lipids (waxes) obtained by chloroform extraction, as well as the monomeric composition of native and wound suberin polymer after transesterification by boron trifluoride/methanol, was analyzed using gas chromatography and mass spectrometry. Both types of periderm contained up to 20% extractable lipids. Besides linear long-chain aliphatic wax compounds, alkyl ferulates were detected as significant constituents. In wound periderm they amounted to more than 60% of the total extracts. Within 1 month of storage, suberin amounts in the polymer increased 2-fold in native periderm (180 g cm^–2), whereas in wound periderm about 75.0 g cm^–2 suberin polymer was newly synthesized. Native potato tuber periderm developed a very efficient transport barrier for water with permeances decreasing from 6.4×10^–10 m s^–1 to 5.5×10^–11 m s^–1 within 1 month of storage. However, the water permeability of wound periderm was on average 100 times higher with permeances decreasing from 4.7×10^–8 m s^–1 after 3 days to only 5.4×10^–9 m s^–1 after 1 month of storage, although suberin and wax amounts in wound periderm amounted to about 60% of native periderm. From this result it must be concluded that the occurrence of suberin with wax depositions in cell walls does not necessarily allow us to conclude that these cell walls must be nearly perfect barriers to water transport. In addition to the occurrence of the lipophilic biopolymer suberin and associated waxes, the still unknown molecular arrangement and precisely localized deposition of suberin within the cell wall must contribute to the efficiency of suberin as a barrier to water transport.     

Induktionsbedingungen der Suberinsynthese und Zellproliferation bei Parenchymfragmenten der Kartoffelknolle

Summary Experiments with potato tuber fragments under normal atmospheric conditions (0.03% CO₂) suggest that a wound periderm, prominently marked by suberin synthesis, develops regularly, independently of preparation technics. It has been demonstrated that potato parenchym is able to perform differentiated reactions depending on the changing influences on the reacting cells caused by the wound stimulus and partial isolation. Suberin synthesis and the cicatrization effect can be suppressed totally if changes remain insignificant as compared with the cellular state in the intact organ. This requires: 1. separating the tissue without bruising it to avoid deformations of the protoplasmatic structure; 2. removing adjacent fragments of destroyed cells by rinsing the slices; 3. maintaining cell turgidity by keeping the moisture in the reaction atmosphere at 100%. Moreover 10% CO₂-concentration is necessary, corresponding to the intercellular air in situ.Whithin 24 hours after depression these conditions induce spontaneous proliferation uniformly in all superficial cell layers of tissue slices, accompanied by multiple mitosis in place of the typical suberized wound periderm. Inhibition of suberin biosynthesis is reversible by subsequent modification of reaction conditions. In contrast a method has been found to induce suberin formation only and to suppress any cell division activity (Kahl et al., 1969 a). This provides the opportunity to analyse causal corresponding physiological reactions in genetically identical cells. The advantage of this method—compared with long term experiments involving hormone-activated growth in sterile tissue culture — is the possibility of controlling quantitative and qualitative changes in biochemical pathways immediately after derepression.

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Diese Arbeit enthält einen Auszug aus der Habilitationsschrift von H. Lange, Naturwissenschaftliche Fakultät der Universität Frankfurt a. M., 1969.     

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.     

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.     

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     

Ultrastructure and chemistry of soluble and polymeric lipids in cell walls from seed coats and fibres of Gossypium species

Electron-microscopic examination in conjunction with extraction procedures and chemical analysis have confirmed that a suberin-like lipid biopolymer is located within the concentric polylamellate layers found in the secondary cell walls of green cotton fibres (Gossypium hirsutum cv. green lint). A polymer of similar ultrastructure and chemical constitution also occurs mainly in the secondary seed-coat walls of the outer epidermis of both green and white varieties of G. hirsutum. The suberins composed of predominantly C₂₂ compounds are, however, markedly different from those present in the periderms of the same plants; these comprise mainly C₁₆ and C₁₈ compounds. Long-chain 1-alkanols (C₂₆–C₃₆) and alkanoic acids (C₁₆–C₃₆) are the principal components of the wax from white fibres but these lipid classes comprise a much smaller proportion of that from green fibres. unidentified highmolecular-weight compounds were the major constituents of the green-fibre was extract which also contains a number of yellow-green pigments, probably flavonoid in nature. These pigments are thought to be associated with the ultrahistochemical reaction with silver proteinate that was observed only in the green-fibre cell walls. A total of 16 wild and cultivated cotton species were examined with the electron microscope for the presence of suberin. The outer seed-coat epidermis of all the examined species but only the fibres of the wild ones were found to be suberized. Among the analysed mutants of fibre colour in G. hirsutum only the gene Lg (green lint) seemed to be associated with suberin.Abbreviations GLC gas-liquid chromatography - TLC thinlayer chromatography Fibres=fibre cells of the seed coat epidermis without fibre base; Seed coast=include the base of fibre cells, and short, so-called fuzz fibres     

Structure, gas chromatographic measurement, and function of suberin synthesized by potato tuber tissue slices

The polymeric material (suberin) of the wound periderm of potato tuber slices was analyzed after depolymerization with LiAIH₄ in tetrahydrofuran or BF₃ in methanol with the use of thin layer chromatography, chemical modification, and combined gas-liquid chromatography and mass spectrometry. Fatty acids (C₁₆ to C₂₆), fatty alcohols (C₁₆ to C₂₆), octadec-9-ene-1, 18-dioic acid, and 18-hydroxy-octadec-9-enoic acid were identified to be the major components. Based on the structural information that the two bifunctional C₁₈ molecules constituted a major portion of suberin, a gas chromatographic method of measuring suberization was developed. This method consisted of hydrogenolysis of powdered tissue followed by thin layer chromatography and gas chromatographic measurement of octadecene-1, 18-diol as the trimethylsilyl ether. With this assay it was shown that the development of resistance to water loss by the tissue slices was directly proportional to the quantity of the bifunctional C₁₈ molecules, thus providing evidence that a function of suberin is prevention of water loss.     

Iron Deficiency Decreases Suberization in Bean Roots through a Decrease in Suberin-Specific Peroxidase Activity

The suberin content of young root parts of iron-deficient and iron-sufficient Phaseolus vulgaris L. cv Prélude was determined. The aliphatic components that could be released from suberin-enriched fractions by LiAID₄ depolymerization were identified by gas chromatography-mass spectrometry. In the normal roots, the major aliphatic components were ω-hydroxy acids and dicarboxylic acids in which saturated C₁₆ and monounsaturated C₁₈ were the dominant homologues. Iron-deficient bean roots contained only 11% of the aliphatic components of suberin found in control roots although the relative composition of the constituents was not significantly affected by iron deficiency. Analysis of the aromatic components of the suberin polymer that could be released by alkaline nitrobenzene oxidation of bean root samples showed a 95% decrease in p-hydroxybenzaldehyde, vanillin, and syringaldehyde under iron-deficient conditions. The inhibition of suberin synthesis in bean roots was not due to a decrease in Fe-dependent ω-hydroxylase activity since normal ω-hydroxylation could be demonstrated, both in vitro with microsomal preparations and in situ by labeling of ω-hydroxy and dicarboxylic acids with [¹⁴C]acetate. The level of the isozyme of peroxidase that is specifically associated with suberization was suppressed by iron deficiency to 25% of that found in control roots. None of the other extracted isozymes of peroxidase was affected by the iron nutritional status. The activity of the suberin-associated peroxidase was restored within 3 to 4 days after application of iron to the growth medium. The results suggest that, in bean roots, iron deficiency causes inhibition of suberization by causing a decrease in the level of isoperoxidase activity which is required for polymerization of the aromatic domains of suberin, while the ability to synthesize the aliphatic components of the suberin polymer is not impaired.     

Suberized bundle sheaths in grasses (Poaceae) of different photosynthetic types I. Anatomy,ultrastructure and histochemistry

Summary We investigated the histochemistry and ultrastructure of the cell walls of mestome sheaths and parenchymatous bundle sheaths of ten species of grasses. The species surveyed included representatives from all the major photosynthetic types: C₃-Bromus tectorum, Phalaris arundinacea; C₄/NAD-ME-Eragrostis cilianensis, Panicum capillare; C₄/NAD-ME/PCK-Bouteloua curtipendula; C₄/PCK-Chloris gayana, Sporobolus elongatus; C₄/NADP-ME-Echinochloa crus-galli, Setaria glauca, Themeda triandra. All vein orders (designated here as major, minor and transverse) from mature leaves of each species were tested histochemically for lipids and phenols, and the majority of species were also examined with the electron microscope. A suberized lamella was detected ultrastructurally in at least some walls of major vein bundle sheath cells of all species examined. These lamellae were also present in some cells associated with the minor veins of the C₃ species and in the minor and transverse veins of the C₄/NADP-ME species. Histochemical tests for lipids and phenols consistently failed to differentiate this layer. Based on these tests, none of the vein orders in any species showed evidence of a Casparian band. In all suberized bundle sheaths, the compound middle lamella between cells with suberin lamellae is modified by the presence of phenols. These did not, however, confer resistance to acid digestion to the cell layer, in contrast to cell layers with Casparian bands. Therefore, although the mestome sheath has some features in common with the root endodermis (i.e. cells with a suberized lamella and thick, cellulosic walls which may be further modified), we could find no substantive anatomical or ultrastructural evidence for the presence of a Casparian band in any of the grass leaves investigated. The significance of these observations is discussed in the context of apoplastic permeability of these walls.     

Differential induction of polar and non‐polar metabolism during wound‐induced suberization in potato (Solanum tuberosum L.) tubers

Wound‐induced suberin deposition involves the temporal and spatial coordination of phenolic and fatty acid metabolism. Phenolic metabolism leads to both soluble metabolites that accumulate as defense compounds as well as hydroxycinnamoyl derivatives that form the basis of the poly(phenolic) domain found in suberized tissue. Fatty acid metabolism involves the biosynthesis of very‐long‐chain fatty acids, 1‐alkanols, ω‐hydroxy fatty acids and α,ω‐dioic acids that form a poly(aliphatic) domain, commonly referred to as suberin. Using the abscisic acid (ABA) biosynthesis inhibitor fluridone (FD), we reduced wound‐induced de novo biosynthesis of ABA in potato tubers, and measured the impact on the expression of genes involved in phenolic metabolism (StPAL1, StC4H, StCCR, StTHT), aliphatic metabolism (StCYP86A33, StCYP86B12, StFAR3, StKCS6), metabolism linking phenolics and aliphatics (StFHT) or acyl chains and glycerol (StGPAT5, StGPAT6), and in the delivery of aliphatic monomers to the site of suberization (StABCG1). In FD‐treated tissue, both aliphatic gene expression and accumulation of aliphatic suberin monomers were delayed. Exogenous ABA restored normal aliphatic suberin deposition in FD‐treated tissue, and enhanced aliphatic gene expression and poly(aliphatic) domain deposition when applied alone. By contrast, phenolic metabolism genes were not affected by FD treatment, while FD + ABA and ABA treatments slightly enhanced the accumulation of polar metabolites. These data support a role for ABA in the differential induction of phenolic and aliphatic metabolism during wound‐induced suberization in potato.     

Biochemistry of Suberization: Incorporation of [1-C]Oleic Acid and [1-C]Acetate into the Aliphatic Components of Suberin in Potato Tuber Disks (Solanum tuberosum)

Biosynthesis of the aliphatic components of suberin was studied in suberizing potato (Solanum tuberosum) slices with [1-¹⁴C]oleic acid and [1-¹⁴C]acetate as precursors. In 4-day aged tissue, [1-¹⁴C]oleic acid was incorporated into an insoluble residue, which, upon hydrogenolysis (LiA1H₄), released the label into chloroform-soluble products. Radio thin layer and gas chromatographic analyses of these products showed that ¹⁴C was contained exclusively in octadecenol and octadecene-1, 18-diol. OsO₄ treatment and periodate cleavage of the resulting tetraol showed that the labeled diol was octadec-9-ene-1, 18-diol, the product expected from the two major components of suberin, namely 18-hydroxyoleic acid and the corresponding dicarboxylic acid. Aged potato slices also incorporated [1-¹⁴C]acetate into an insoluble material. Hydrogenolysis followed by radio chromatographic analyses of the products showed that ¹⁴C was contained in alkanols and alkane-α,ω-diols. In the former fraction, a substantial proportion of the label was contained in aliphatic chains longer than C₂₀, which are known to be common constituents of suberin. In the labeled diol fraction, the major component was octadec-9-ene-1,18-diol, with smaller quantities of saturated C₁₆, C₁₈, C₂₀, C₂₂, and C₂₄-α,ω-diols. Soluble lipids derived from [1-¹⁴C]acetate in the aged tissue also contained labeled very long acids from C₂₀ to C₂₈, as well as C₂₂ and C₂₄ alcohols, but no labeled ω-hydroxy acids or dicarboxylic acids were detected. Label was also found in n-alkanes isolated from the soluble lipids, and the distribution of label among them was consistent with the composition of n-alkanes found in the wound periderm of this tissue; C₂₁ and C₂₃ were the major components with lesser amounts of C₁₉ and C₂₅. The amount of ¹⁴C incorporated into these bifunctional monomers in 0-, 2-, 4-, 6-, and 8-day aged tissue were 0, 1.5, 2.5, 0.8, and 0.3% of the applied [1-¹⁴C]oleic acid, respectively. Incorporation of [1-¹⁴C]acetate into the insoluble residue was low up to the 3rd day of aging, rapid during the next 4 days of aging, and subsequently the rate decreased. These changes in the rates of incorporation of exogenous oleic acid and acetate reflected the development of diffusion resistance of the tissue surface to water vapor. As the tissue aged, increasing amounts of the [1-¹⁴C]acetate were incorporated into longer aliphatic chains of the residue and the soluble lipids, but no changes in the distribution of radioactivity among the α-ω-diols were obvious. The above results demonstrated that aging potato slices constitute a convenient system with which to study the biochemistry of suberization.     

The poly(phenolic) domain of potato suberin: a non-lignin cell wall bio-polymer.

Suberized plant cell walls have three distinguishing features: (1) tissue specificity, (2) a poly(aliphatic) domain and (3) a unique, "lignin-like" poly(phenolic) domain. With respect to the latter, comparisons have often been made to lignin, but the unique phenolic composition of suberized cells yields a unique polymer better designated as a poly(phenolic) domain. Potato tubers that have been induced to suberize through wounding make an excellent model system with which the chemistry, biochemistry and macromolecular assembly of the suberin poly(phenolic) domain can be monitored. For example, wound healing potato tubers have been used to determine the unique hydroxycinnamic acid nature of its poly(phenolic) domain using specific carbon-13 labeling studies and specific chemical degradation techniques (e.g. thioacidolysis). Furthermore, a suberization-associated anionic peroxidase has been purified from suberizing potato tubers and subsequently shown to oxidize hydroxycinnamic acids (and their derivatives) in preference to monolignols, as well as yield an unique polymer in vitro. We have since extended these studies to begin analyzing the macromolecular assembly process leading to the deposition of this suberized tissue specific domain. To this end we have begun to describe an H(2)O(2)-generating system with NAD(P)H-dependent oxidase-like properties that is temporally associated with the formation of potato suberin poly(phenolics) during suberization. Herein we describe our progress to date.     

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.     

Chemical Composition and Ultrastructure of Suberin from Hollow Heart Tissue of Potato Tubers (Solanum tuberosum)

The disorder of potato tubers (Solanum tuberosum var. Russet Burbank) called “hollow heart” is manifested by the occurrence of hollow regions in internal parts of the tuber. The structure and composition of the suberin from the tissue lining of these internal cavities were determined by gas chromatography and mass spectrometry of the LiAlH₄-hydrogenolysis products. Identification of octadecene-1,18-diol as the major component and the presence of hexadecane-1,16-diol and very long chain (>C₁₈) alcohols in the hydrogenolysate showed that the suberin lining the internal cavities is quite similar to that found in the periderm of external wounds and the natural skin. Electron microscopic examination showed similar lamellar structure for the suberin of hollow heart, external wound periderm, and the natural skin of potato tubers. The results show that suberin can develop in a tissue which is not exposed to the external environment.