Biochemical characterization of the suberization-associated anionic peroxidase of potato.

The anionic peroxidase associated with the suberization response in potato (Solanum tuberosum L.) tubers during wound healing has been purified and partially characterized at the biochemical level. It is a 45-kD, class III (plant secretory) peroxidase that is localized to suberizing tissues and shows a preference for feruloyl (o-methoxyphenol)-substituted substrates (order of substrate preference: feruloyl > caffeoyl > p-coumaryl approximately syringyl) such as those that accumulate in tubers during wound healing. There was little influence on oxidation by side chain derivatization, although hydroxycinnamates were preferred over the corresponding hydroxycinnamyl alcohols. The substrate specificity pattern is consistent with the natural substrate incorporation into potato wound suberin. In contrast, the cationic peroxidase(s) induced in response to wound healing in potato tubers is present in both suberizing and nonsuberizing tissues and does not discriminate between hydroxycinnamates and hydroxycinnamyl alcohols. A synthetic polymer prepared using E-[8-(13)C]ferulic acid, H(2)O(2), and the purified anionic enzyme contained a significant amount of cross-linking through C-8, albeit with retention of unsaturation.     

Wound-Induced Metabolism in Potato (Solanum tuberosum) Tubers: Biosynthesis of Aliphatic Domain Monomers

Suberin, a cell specific, wall-associated biopolymer, is formed during normal plant growth and development as well as in response to stress conditions such as wounding. It is characterized by the deposition of both a poly(phenolic) domain (SPPD) in the cell wall and a poly(aliphatic) domain (SPAD) thought to be deposited between the cell wall and plasma membrane. Although the monomeric components that comprise the SPPD and SPAD are well known, the biosynthesis and deposition of suberin is poorly understood. Using wound healing potato tubers as a model system, we have tracked the flux of carbon into the aliphatic monomers of the SPAD in a time course fashion. From these analyses, we demonstrate that newly formed fatty acids undergo one of two main metabolic fates during wound-induced suberization: (1) desaturation followed by oxidation to form the 18:1 ω-hydroxy and dioic acids characteristic of potato suberin, and (2) elongation to very long chain fatty acids (C20 to C28), associated with reduction to 1-alkanols, decarboxylation to n-alkanes and minor amounts of hydroxylation. The partitioning of carbon between these two metabolic fates illustrates metabolic regulation during wound healing, and provides insight into the organization of fatty acid metabolism.Key Words: suberin, potato, Solanum tuberosum, carbon flux analysis, abiotic stress     

Biosynthesis, deposition, and partial characterization of potato suberin phenolics

Alkaline nitrobenzene oxidation of the polymeric materials from wound-healed potato (Solanum tuberosum L. var. White Rose) tuber tissue liberated p-hydroxybenzaldehyde, vanillin, and minor amounts of syringaldehyde as determined by gas chromatography/mass spectrometry. The aromatic aldehydes were derived only from periderm. The amounts of aromatic aldehydes liberated were used as a measure of the deposition of phenolic suberin components. Phenolic deposition began after about 2 days of wound healing; after 8 days the amounts of p-hydroxybenzaldehyde released by nitrobenzene oxidation leveled off at 5 milligrams per gram dry weight and after 12 days vanillin liberation reached a maximum at 7.5 milligrams per gram dry weight. The time course of deposition of the phenolic polymeric material is analogous to that reported for the deposition of the aliphatic components of suberin and therefore these results are consistent with the proposed structure of suberin. Experiments with radiolabeled l-phenylalanine and cinnamic acid indicated that exogenous phenylalanine was less efficient than cinnamic acid as a precursor of suberin phenolics. Nitrobenzene oxidation of radiolabeled suberin preparations gave three major labeled fractions: a diethyl ether-soluble fraction containing aromatic aldehydes ( approximately 20%), an ethyl acetate-soluble fraction containing unknown compounds ( approximately 15%), and a condensed phenolic fraction ( approximately 10%). Thin-layer and gas-liquid chromatographic analysis of the ether fraction showed that the major labeled components were vanillin and p-hydroxybenzaldehyde. The condensed tannin fraction revealed the presence of several labeled macromolecular phenolic fractions. Elution profiles of the condensed tannin fraction from tissues suberized for different periods of time were essentially identical, suggesting qualitative similarity of deposition and polymerization of suberin phenolics throughout the duration of wound healing. Chlorogenic acid accumulation in wound healing potato tuber discs was measured by high-performance liquid chromatography. The level of this compound reached 130 micrograms per disk after 11 days and did not decline even after the deposition of suberin ceased, revealing no precursor role for this acid in suberization.     

Catalase inhibition alters suberization and wound healing in potato (Solanum tuberosum) tubers

In response to wounding, potato ( Solanum tuberosum L.) tubers generate hydrogen peroxide (H₂O₂) in association with suberization, a critical phase of the wound-healing process. In the present study, the effect of aminotriazole (AT), a catalase (CAT, EC 1.11.1.6) inhibitor, on cut tubers was investigated using fresh weight (FW) loss and pathogen attack symptoms as indicators of wound-healing efficiency. Seven days after treatment, AT-treated tuber halves lost more FW and developed infection signs compared with the controls. Thiourea, another CAT inhibitor, as well as exogenous H₂O₂ treatments induced the same effects as AT suggesting that the alteration of the wound healing may be caused by CAT inhibition and the resulting accumulation of H₂O₂. Using transgenic tubers, FW losses 1 week after wounding were either higher (CAT repression) or lower (CAT overexpression) than those of the wild-type. When tuber halves were allowed to wound heal for different periods before treatment, AT had no effect on the progress of their wound healing if wound-healed for at least 3 days. This implies that AT may affect early wound-healing-related events, especially those occurring before or during suberization. A time-course analysis of the effects of AT treatment on wounded tuber tissues revealed that AT prevented the deposition of the polyphenolic domain of suberin in association with CAT inhibition and H₂O₂ accumulation. These data are important in identifying factors that may be required to regulate suberization and contribute to a better understanding of this critical process to hasten its rate and limit wound-related losses in stored potato tubers.     

A feruloyltyramine trimer isolated from potato common scab lesions

(1)H NMR analysis established that a potential suberin intermediate isolated from potato common scab lesions contained three O-methyl groups, a phenylcoumaran-type linkage and a conjugated trans double bond. Mass spectral data determined its molecular formula as indicative of a dehydrotrimer structure formed from three feruloyltyramine units. (1)H and (13)C NMR correlation studies supported the structure as that of a grossamide unit (3) linked through its double bond to the feruloyl phenolic of a third feruloyltyramine group. Identification of the feruloyltyramine trimer (4) expands the number of cross-linked intermediates potentially involved in the suberization process and highlights the presence of a second type of inter-unit linkage available for synthesis of the poly-phenolic domains.     

CHLOROGENIC ACID ACCUMULATION AND WOUND HEALING IN SWEET POTATO ROOTS

Mc Clure , T. T. (Plant Pest Control Division, ARS, USDA, Washington, D. C.) Chlorogenic acid accumulation and wound healing in sweet potato roots . Amer. Jour. Bot. 47(4) : 277—280. Illus. 1960.–Chlorogenic acid accumulation in cells adjacent to a wound occurs before suberization and wound-periderm formation. Suberization during wound healing was highly correlated with chlorogenic acid accumulation and with wound-periderm formation. The possible role of chlorogenic acid as a source of chemical units for suberization is suggested. Histochemical tests indicate that suberization during wound healing may be a form of lignification. Over 5 times as much lignin was found by chemical analysis in the tissues of healed surfaces as in controls.     

Regulatory involvement of abscisic acid in potato tuber wound-healing

Rapid wound-healing is crucial in protecting potato tubers frominfection and dehydration. Wound-induced suberization and theaccumulation of hydrophobic barriers to reduce water vapourconductance/loss are principal protective wound-healing processes.However, little is known about the cognate mechanisms that effector regulate these processes. The objective of this researchwas to determine the involvement of abscisic acid (ABA) in theregulation of wound-induced suberization and tuber water vapourloss (dehydration). Analysis by liquid chromatography–massspectrometry showed that ABA concentrations varied little throughoutthe tuber, but were slightly higher near the periderm and lowestin the pith. ABA concentrations increase then decrease duringtuber storage. Tuber wounding induced changes in ABA content.ABA content in wound-healing tuber discs decreased after wounding,reached a minimum by 24 h, and then increased from the 3rd tothe 7th day after wounding. Wound-induced ABA accumulationswere reduced by fluridone (FLD); an inhibitor of de novo ABAbiosynthesis. Wound-induced phenylalanine ammonia lyase activitywas slightly reduced and the accumulation of suberin poly(phenolics)and poly(aliphatics) noticeably reduced in FLD-treated tissues.Addition of ABA to the FLD treatment restored phenylalanineammonia lyase activity and suberization, unequivocally indicatingthat endogenous ABA is involved in the regulation of these wound-healingprocesses. Similar experiments showed that endogenous ABA isinvolved in the regulation of water vapour loss, a process linkedto wax accumulation in wound-healing tubers. Rapid reductionof water vapour loss across the wound surface is essential inpreventing desiccation and death of cells at the wound site;live cells are required for suberization. These results unequivocallyshow that endogenous ABA is involved in the regulation of wound-inducedsuberization and the processes that protect surface cells fromwater vapour loss and death by dehydration. Key words: Abscisic acid, poly(aliphatic), poly(phenolic), potato, Solanum tuberosum L., suberin     

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.     

Reactive oxygen species production in association with suberization: evidence for an NADPH-dependent oxidase

In response to wounding, potato tubers generate reactive oxygen species (ROS) in association with suberization. Immediately following wounding, an initial burst of ROS occurs, reaching a maximum within 30 to 60 min. In addition to this initial oxidative burst, at least three other massive bursts occur at 42, 63 and 100 h post-wounding. These latter bursts are associated with wound healing and are probably involved in the oxidative cross-linking of suberin poly(phenolics). The source of ROS is likely to be a plasma membrane NADPH-dependent oxidase immunorelated to the human phagocyte plasma membrane oxidase. The initial oxidative burst does not appear to be dependent on new protein synthesis, but the subsequent bursts are associated with an increase in oxidase protein components. Oxidase activity is enhanced in vitro by hydroxycinnamic acids and conjugates associated with the wound healing response in potato.     

Metabolite profiling of potato (Solanum tuberosum L.) tubers during wound-induced suberization

Suberin is a specific cell wall-associated biopolymer characterized by the deposition of both a poly(phenolic) domain (SPPD) associated with the cell wall, and a poly(aliphatic) domain (SPAD) thought to be deposited between the cell wall and plasma membrane. In planta, suberin functions to prevent plants from desiccation and pathogen attack. Although the chemical identity of the monomeric components of the SPPD and SPAD are well known, their concerted biosynthesis and assembly into the suberin macromolecule is poorly understood. To expand our knowledge of suberin biosynthesis, a GC/MS-based metabolite profiling study was conducted, using wound healing potato (Solanum tuberosum L.) tubers as a model system. A time series of both non-polar and polar metabolite profiles were created, yielding a broad-based, dynamic picture of wound-induced metabolism, including suberization. Principal component analysis revealed a separation of metabolite profiles according to different suberization stages, with clear temporal differences emerging in the non-polar and polar profiles. In the non-polar profiles, suberin-associated aliphatics contributed the most to cluster formation, while a broader range of metabolites (including organic acids, sugars, amino acids and phenylpropanoids) influenced cluster formation amongst polar profiles. Pair-wise correlation analysis revealed strong correlations between known suberin-associated compounds, as well as between suberin-associated compounds and several un-identified metabolites in the profiles. These data may help to identify additional, as yet unknown metabolites associated with suberization process.     

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.     

Changes in flux pattern of the central carbohydrate metabolism during kernel development in maize

Developing kernels of the inbred maize line W22 were grown in sterile culture and supplied with a mixture of [U-13C6]glucose and unlabeled glucose during three consecutive intervals (11-18, 18-25, or 25-32 days after pollination) within the linear phase of starch formation. At the end of each labeling period, glucose was prepared from starch and analyzed by 13C isotope ratio mass spectrometry and high-resolution (13)C NMR spectroscopy. The abundances of individual glucose isotopologs were calculated by computational deconvolution of the NMR data. [1,2-(13)C2]-, [5,6-(13)C2]-, [2,3-(13)C2]-, [4,5-(13)C2]-, [1,2,3-(13)C3]-, [4,5,6-(13)C3]-, [3,4,5,6-(13)C4]-, and [U-(13)C6]-isotopologs were detected as the major multiple-labeled glucose species, albeit at different normalized abundances in the three intervals. Relative flux contributions by five different pathways in the primary carbohydrate metabolism were determined by computational simulation of the isotopolog space of glucose. The relative fractions of some of these processes in the overall glucose cycling changed significantly during maize kernel development. The simulation showed that cycling via the non-oxidative pentose phosphate pathway was lowest during the middle interval of the experiment. The observed flux pattern could by explained by a low demand for amino acid precursors recruited from the pentose phosphate pathway during the middle interval of kernel development.     

Physiological Basis for a Cycloheximide-induced Soft Rot of Potatoes by Pseudomonas fluorescens

Cycloheximide renders discs of potato tissue (Solanum tuberosum,cultivar Kennebec) susceptible to soft rot by a non-pathogenicisolate of Pseudomonas fluorescens. Pectate lyases (E.C. 4.2.99.3 [EC] )are the dominant extracellular macerating agents produced bythe test organism. Potato discs aged 24 h become resistant tomaceration by purified lyase preparations. Cycloheximide blocksthe development of resistance by inhibiting suberization. Thesite of inhibition is thought to be the cycloheximide-sensitivesynthesis of phenylalanine ammonia-lyase (E.C. 4.3.1.5 [EC] ) in potatodiscs. This enzyme is necessary for production of phenolic precursorsof suberin. Comparison of tissue from a number of potato cultivarscorrelates the synthesis of phenylalanine ammonia-lyase withresistance of discs to attack by the Pseudomonad. Resistance of potato tissue to pectate lyase is also affectedby intrinsic reactions not involving suberization. Resistanceincreases in fresh unsuberized discs when tubers are transferredfrom cold storage to room temperature before use. Resistancedecreases rapidly when tubers are transferred back to the cold.The intrinsic resistance appears to increase in the surfacelayer of cells in ageing discs. It is estimated that intrinsicreactions and suberization contribute equally to resistanceof aged discs to pectate lyase maceration.     

Validation of deuterium-labeled fatty acids for the measurement of dietary fat oxidation during physical activity

Measurement of 13C-labeled fatty acid oxidation is hindered by the need for acetate correction, measurement of the rate of CO2 production in a controlled environment, and frequent collection of breath samples. The use of deuterium-labeled fatty acids may overcome these limitations. Herein, d31-palmitate was validated against [1-13C]palmitate during exercise. Thirteen subjects with body mass index of 22.9 +/- 3 kg/m2 and body fat of 19.6 +/- 11% were subjected to 2 or 4 h of exercise at 25% maximum volume oxygen consumption (VO2max). The d31-palmitate and [1-13C] palmitate were given orally in a liquid meal at breakfast. The d3-acetate and [1-13C]acetate were given during another visit for acetate sequestration correction. Recovery of d31-palmitate in urine at 9 h after dose was compared with [1-13C] palmitate recovery in breath. Cumulative recovery of d31-palmitate was 10.6 +/- 3% and that of [1-13C]palmitate was 5.6 +/- 2%. The d3-acetate and [1-13C]acetate recoveries were 85 +/- 4% and 54 +/- 4%, respectively. When [1-13C]acetate recovery was used to correct 13C data, the average recovery differences were 0.4 +/- 3%. Uncorrected d31-palmitate and acetate-corrected [1-13C]palmitate were well correlated (y=0.96x + 0; P <0.0001) when used to measure fatty acid oxidation during exercise. Thus, d31-palmitate can be used in outpatient settings as it eliminates the need for acetate correction and frequent sampling.     

Cerebral metabolism of [1,2-13C2]acetate as detected by in vivo and in vitro 13C NMR

The metabolism of [1,2-13C2]acetate in rat brain was studied by in vivo and in vitro 13C NMR spectroscopy, in particular by taking advantage of the homonuclear 13C-13C spin coupling patterns. Well nourished rats were infused with [1,2-13C2]acetate or [1-13C]acetate in the jugular vein, and the in situ kinetics of 13C labeling during the infusion period was followed by 13C NMR techniques. The in vivo 13C NMR spectra showed signals from (i) the C-1 carbon of [1,2-13C2] acetate or [1-13C]acetate, (ii) 13CO3H-, and (iii) the natural abundance 13C carbons of sufficiently mobile fatty acids. Methanol/HCl/perchloric acid extracts of the brains were prepared and were further analyzed by high resolution 13C NMR. The homonuclear 13C-13C spin coupling patterns after infusion of [1,2-13C2]acetate showed very different isotopomer populations in glutamate, glutamine, and gamma-aminobutyric acid. Analyzing the relative proportions of these isotopomers revealed (i) two different glutamate compartments in the rat brain characterized by the presence and absence, respectively, of glutamine synthase activity, (ii) two different tricarboxylic acid cycles, one preferentially metabolizing [(1,2-13C2]acetate, the other mainly using unlabeled acetyl-coenzyme A, (iii) a hitherto unknown cerebral pyruvate recycling system associated with the tricarboxylic acid cycle, metabolizing primarily unlabeled acetyl-coenzyme A, and (iv) a predominant production of gamma-aminobutyric acid in the glutamate compartment lacking glutamine synthase.     

C-methylation occurs during the biosynthesis of heme d1

The biosynthetic origin of methyl groups in heme d1 isolated from the nitrite reductase cytochrome cd1 was investigated by a stable isotope labeling experiment. Pseudomonas aeruginosa (American Type Culture Collection strain 19429) was grown on a minimal medium supplemented with [13C]methionine. The enzyme was purified, the heme extracted, converted into the free base methyl ester derivative, and purified. 1H NMR and 13C NMR indicated that only the methyl groups attached to C2 and C7 are derived from methionine.