Wound-healing Potato Tuber Research Articles

Transcriptomic analysis of wound-healing in Solanum tuberosum (potato) tubers: Evidence for a stepwise induction of suberin-associated genes

Suberin deposition involves both phenolic and aliphatic polymer biosynthesis and deposition in the same tissue. Therefore, any consideration of exploiting suberin for crop enhancement (e.g., enhanced storage, soil borne disease resistance) requires knowledge of both phenolic and aliphatic component biosynthesis and their coordinated, temporal deposition. In the present study, we use a wound-healing potato tuber system to explore global transcriptome changes during the early stages of wound-healing. Wounding leads to initial and substantial transcriptional changes that follow distinctive temporal patterns – primary metabolic pathways were already functional, or up-regulated immediately, and maintained at levels that would allow for precursor carbon skeletons and energy to feed into downstream metabolic processes. Genes involved in pathways for phenolic production (i.e., the shikimate pathway and phenylpropanoid metabolism) were up-regulated early while those involved in aliphatic suberin production (i.e., fatty acid biosynthesis and modification) were transcribed later into the time course. The pattern of accumulation of genes associated with ABA biosynthesis and degradation steps support a role for ABA in regulating aliphatic suberin production. Evaluation of putative Casparian strip membrane-like genes pinpointed wound-responsive candidates that may mediate the suberin deposition process.

Isolation and Identification of Triglycerides and Ester Oligomers from Partial Degradation of Potato Suberin

Suberized cell walls from wound-healing potato tubers ( Solanum tuberosum ) were depolymerized under mild conditions using methanolic potassium hydroxide in order to investigate the chemical linkages present in this protective plant biopolymer. Analysis of the resulting soluble oligomeric fragments with HPLC, 1D and 2D NMR, LC/MS, and MS(n) methods allowed identification of several novel compounds: a family of homologous triglycerides, a family of homologous aliphatic ester trimers, and an ether-linked phenylacetic acid dimer. These findings illustrate the diversity of rigid and flexible molecular linkages present in both poly(aliphatic) and poly(aromatic) domains of potato suberin, and they point toward architectures that may account for its function as a potent hydrophobic barrier to water, thermal equilibration, and microbial pathogens.

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.

Wound-Induced Metabolism in Potato (Solanum tuberosum) Tubers

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.

Metabolic flux analysis of the phenylpropanoid pathway in wound-healing potato tuber tissue using stable isotope-labeled tracer and LC-MS spectroscopy.

The metabolic flux of two phenylpropanoid metabolites, N-p-coumaroyloctopamine (p-CO) and chlorogenic acid (CGA), in the wound-healing potato tuber tissue was quantitatively analyzed by a newly developed method based upon the tracer experiment using stable isotope-labeled compounds and LC-MS. Tuber disks were treated with aqueous solution of L-phenyl-d(5)-alanine, and the change in the ratio of stable isotope-labeled compound to non-labeled (isotope abundance) was monitored for p-CO and CGA in the tissue extract by LC-MS. The time-dependent change in the isotope abundance of each metabolite was fitted to an equation that was derived from the formation and conversion kinetics of each compound. Good correlations were obtained between the observed and calculated isotope abundances for both p-CO and CGA. The rates of p-CO formation and conversion (i.e. fluxes) were 1.15 and 0.96 nmol (g FW)(-1) h(-1), respectively, and for CGA, the rates 4.63 and 0.42 nmol (g FW)(-1) h(-1), respectively. This analysis enabled a direct comparison of the biosynthetic activity between these two compounds.

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 2O 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.

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 alpha,omega-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.

Purification and characterization of hydroxycinnamoyl-coenzyme A: ?-hydroxypalmitic acid O-hydroxycinnamoyltransferase from tobacco (Nicotiana tabacum L.) cell-suspension cultures

An acyltransferase hydroxycinnamoyl-Coenzyme A: ω-hydroxypalmitic acid O-hydroxycinnamoyltransferase (HHT; EC 2.3.1.-), which transfers hydroxycinnamic acids from hydroxycinnamoyl-CoA thioesters to several hydroxylated fatty acid derivatives, was characterized from tobacco (Nicotiana tabacum L. cv. Xanthi nc) cell-suspension cultures. It exhibited the same properties as the enzyme previously detected in wound-healing potato tuber discs (Lotfy et al., 1994, Phytochemistry 35: 1419–1424), and especially a marked specificity for ω-hydroxypalmitic acid and feruloyl-CoA. It was purified 300-fold to near homogeneity from late logarithmic-phase cell suspensions. The apparent molecular mass of the native protein was 55 kDa and its isoelectric point, estimated by electrofocusing, was 4.6. The purified enzyme conjugated ferulic acid to ω-hydroxypalmitic acid and to 1-tetradecanol, its main lipidic substrates, suggesting that the same enzyme probably synthesizes the different esters of 1-alkanols and of ω-hydroxy fatty acids which are formed in vitro.

Modulation of the Activity of two Hydroxycinnamoyl Transferases in Wound-Healing Potato Tuber Discs in Response to Pectinase or Abscisic Acid

Summary The activity of two enzymes known to be involved in the biosynthesis of cell-wall phenolics in wound-healing potato tuber tissues was monitored in tuber discs treated with abscisic acid (ABA), pectinase, arachidonic acid, or jasmonic acid. Tyramine hydroxycinnamoyl transferase (THT) activity was strongly stimulated by pectinase and, to a lower extent, by ABA. By contrast, ω-hydroxypalmitic acid hydroxycinnamoyl transferase (HHT), which is a good marker of suberin biosynthesis, was induced by ABA, but not by pectinase treatments. Neither arachidonic acid, a known elicitor of phytoalexin biosynthesis in potato tubers, nor jasmonic acid, a putative intermediate in the signal transduction to wounding, significantly modified the time course or the intensity of induction of THT or HHT activities.

Formation of ω-feruloyloxypalmitic acid by an enzyme from wound-healing potato tuber discs

An enzyme, extracted from the outermost cell layers of wound-healing potato tuber discs, has been shown to transfer ferulic acid from feruloyl-CoA to ω-hydroxypalmitic acid. p-Coumaroyl-CoA and sinapoyl-CoA also acted as substrates but were less active than feruloyl-CoA. Good correlation was found between the development of the enzymic activity and the accumulation of alkali-soluble ferulic acid in the suberized cell walls. This transferase (hydroxycinnamoyl-CoA: ω-hydroxypalmitic acid O-hydroxycinnamoyltransferase, HHT, EC 2.3.1.—) could be involved in suberin biosynthesis. Several 1-alkanols were also conjugated to ferulic acid in crude enzymic extracts of wound-healing potato tuber discs. Maximal activity was obtained with 1-tetradecanol, suggesting that ω-hydroxypalmitic acid and 1-tetradecanol could be conjugated by distinct enzymes.

Alkyl ferulates in wound healing potato tubers

Seven ferulic acid esters of 1-alkanols ranging in carbon length from C 16 to C 21 were synthesized and an HPLC protocol for their separation developed. Extracts prepared from wound healing potato ( Solanum tuberosum) tubers and analysed by HPLC indicated that alkyl ferulate esters begin to accumulate 3–7 days after wound treatment. Of the nine esters identified by EIMS, (including two esters of odd chain length alkanols) hexadecyl and octadecyl ferulates were predominant. Alkyl ferulate esters were restricted to the wound periderm.

Molecular cloning, nucleotide sequence, and abscisic acid induction of a suberization-associated highly anionic peroxidase

A highly anionic peroxidase induced in suberizing cells was suggested to be the key enzyme involved in polymerization of phenolic monomers to generate the aromatic matrix of suberin. The enzyme encoded by a potato cDNA was found to be highly homologous to the anionic peroxidase induced in suberizing tomato fruit. A tomato genomic library was screened using the potato anionic peroxidase cDNA and one genomic clone was isolated that contained two tandemly oriented anionic peroxidase genes. These genes were sequenced and were 96% and 87% identical to the mRNA for potato anionic peroxidase. Both genes consist of three exons with the relative positions of their two introns being conserved between the two genes. Primer extension analysis showed that only one of the genes is expressed in the periderm of 3 day wound-healed tomato fruits. Southern blot analyses suggested that there are two copies each of the two highly homologous genes per haploid genome in both potato and tomato. Abscisic acid (ABA) induced the accumulation of the anionic peroxidase transcripts in potato and tomato callus tissues. Northern blots showed that peroxidase mRNA was detectable at 2 days and was maximal at 8 days after transfer of potato callus to solid agar media containing 10(-4) M ABA. The transcripts induced by ABA in both potato and tomato callus were identical in size to those induced in wound-healing potato tuber and tomato fruit. The anionic peroxidase peptide was detected in extracts of potato callus grown on the ABA-containing media by western blot analysis. The results support the suggestion that stimulation of suberization by ABA involves the induction of the highly anionic peroxidase.

Immunocytochemical Localization and Time Course of Appearance of an Anionic Peroxidase Associated with Suberization in Wound-Healing Potato Tuber Tissue

Thin sections of wound-healing potato tuber tissue were stained with rabbit antibody prepared against a suberization-associated anionic peroxidase and then stained with a goat anti-rabbit antibody-fluorescein conjugate. When these sections were examined with an epiilluminating fluorescence microscope, bright green fluorescent linear deposits were observed on the inner side of cell walls in the periderm layer. Initial deposits which were often not contiguous throughout the wall were first observed in some cells after 3 days of wound-healing and subsequently these layers became more pronounced so that all 6 day old periderm cells had green fluorescent layers on their inner walls. This fluorescence was not present in the walls of parenchyma cells or in the walls of periderm cells treated with preimmune serum and anti-rabbit IgG-FITC conjugate. Thin sections of wound-healing potato tissue which were stained with anti-peroxidase antibody and a goat anti-rabbit antibody-rhodamine conjugate exhibited a similar time course of development with a bright reddish-orange fluorescent layer observed on the inside wall of periderm cells. The production of this suberization-associated anionic peroxidase in wound-healing tissue was also demonstrated by an immunobinding dot blot assay which showed that the largest increase in the enzyme level occurred between 4 and 6 days of wound-healing. The present results support the hypothesis that this anionic peroxidase is involved in the deposition of the aromatic polymeric domain of suberin.

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.

Purification and characterization of a wound-induced ω-hydroxyfatty acid:NADP oxidoreductase from potato tuber disks ( Solanum tuberosum L.)

ω-Hydroxyfatty acid dehydrogenase (ω-hydroxyfatty acid:NADP oxidoreductase) catalyzes the reaction ω-hydroxyfatty acid + NADP ⇌ ω-oxofatty acid + NADPH +H +. In wound-healing potato tuber disks, the ω-oxofatty acid generated by this enzyme is further oxidized to the corresponding dicarboxylic acid by a separate enzyme, ω-oxofatty acid dehydrogenase. ω-Hydroxy acid dehydrogenase, but not ω-oxo acid dehydrogenase, was found to be induced by wounding potato tubers. ω-Hydroxy acid dehydrogenase has been purified 600-fold to near homogeneity from wound-healing potato tuber disks by a combination of gel filtration, anion-exchange, and hydroxylapatite chromatography followed by NADP-Sepharose affinity chromatography, in about 1% yield. The molecular weight and Stokes radius of this enzyme as determined by gel exclusion chromatography are 60,000 and 31 Å, respectively. Sodium dodecyl sulfate-gel electrophoresis gave a molecular weight of 31,000, indicating that the deydrogenase is a dimer with subunits of similar molecular weight. The pH optima for the reaction in the forward and reverse directions are 9.5 and 8.5, respectively, and V in the forward and reverse directions are 140 and 3200 nmol/min/mg, respectively. Apparent K m values for NADP, 16-hydroxyhexadecanoic acid, NADPH, and 16-oxohexadecanoic acid are 100, 20, 5, and 7 μ m respectively. The equilibrium constant of the reaction at pH 9.5 and 30 °C is 1.4 × 10 −9 m. The enzyme preparation did not show any stereospecificity for hydride transfer from NADPH to 16-oxohexadecanoic acid.