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.     

Perturbation of polyamine catabolism can strongly affect root development and xylem differentiation

Spermidine (Spd) treatment inhibited root cell elongation, promoted deposition of phenolics in cell walls of rhizodermis, xylem elements, and vascular parenchyma, and resulted in a higher number of cells resting in G(1) and G(2) phases in the maize (Zea mays) primary root apex. Furthermore, Spd treatment induced nuclear condensation and DNA fragmentation as well as precocious differentiation and cell death in both early metaxylem and late metaxylem precursors. Treatment with either N-prenylagmatine, a selective inhibitor of polyamine oxidase (PAO) enzyme activity, or N,N(1)-dimethylthiourea, a hydrogen peroxide (H(2)O(2)) scavenger, reverted Spd-induced autofluorescence intensification, DNA fragmentation, inhibition of root cell elongation, as well as reduction of percentage of nuclei in S phase. Transmission electron microscopy showed that N-prenylagmatine inhibited the differentiation of the secondary wall of early and late metaxylem elements, and xylem parenchymal cells. Moreover, although root growth and xylem differentiation in antisense PAO tobacco (Nicotiana tabacum) plants were unaltered, overexpression of maize PAO (S-ZmPAO) as well as down-regulation of the gene encoding S-adenosyl-l-methionine decarboxylase via RNAi in tobacco plants promoted vascular cell differentiation and induced programmed cell death in root cap cells. Furthermore, following Spd treatment in maize and ZmPAO overexpression in tobacco, the in vivo H(2)O(2) production was enhanced in xylem tissues. Overall, our results suggest that, after Spd supply or PAO overexpression, H(2)O(2) derived from polyamine catabolism behaves as a signal for secondary wall deposition and for induction of developmental programmed cell death.     

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.     

Hydrogen peroxide acts as a second messenger for the induction of defense genes in tomato plants in response to wounding, systemin, and methyl jasmonate

The systemic accumulation of both hydrogen peroxide (H(2)O(2)) and proteinase inhibitor proteins in tomato leaves in response to wounding was inhibited by the NADPH oxidase inhibitors diphenylene iodonium (DPI), imidazole, and pyridine. The expression of several defense genes in response to wounding, systemin, oligosaccharides, and methyl jasmonate also was inhibited by DPI. These genes, including those of four proteinase inhibitors and polyphenol oxidase, are expressed within 4 to 12 hr after wounding. However, DPI did not inhibit the wound-inducible expression of genes encoding prosystemin, lipoxygenase, and allene oxide synthase, which are associated with the octadecanoid signaling pathway and are expressed 0.5 to 2 hr after wounding. Accordingly, treatment of plants with the H(2)O(2)-generating enzyme glucose oxidase plus glucose resulted in the induction of only the later-expressed defensive genes and not the early-expressed signaling-related genes. H(2)O(2) was cytochemically detected in the cell walls of vascular parenchyma cells and spongy mesophyll cells within 4 hr after wounding of wild-type tomato leaves, but not earlier. The cumulative results suggest that active oxygen species are generated near cell walls of vascular bundle cells by oligogalacturonide fragments produced by wound-inducible polygalacturonase and that the resulting H(2)O(2) acts as a second messenger for the activation of defense genes in mesophyll cells. These data provide a rationale for the sequential, coordinated, and functional roles of systemin, jasmonic acid, oligogalacturonides, and H(2)O(2) signals for systemic signaling in tomato plants in response to wounding.     

Systemic induction of H2O2 in pea seedlings pretreated by wounding and exogenous jasmonic acid

Mechanical stress was one of stresses with whichplants often met. With the development of fruit andvegetable finish machining in food industry, artificialinjury also appeared. As response to other stresses,plants have evolved with some adaptive mechanismsto cope with wounding[1]. Jasmonic acid (JA) andmethyl jasmonate (MeJA), as important signal mole-cules in plant response to wounding, have attracted agreat deal of attention. The studies on some crops, suchas potato[2], rice[3], and tomato[…     

Nitric oxide negatively modulates wound signaling in tomato plants

Synthesis of proteinase inhibitor I protein in response to wounding in leaves of excised tomato (Lycopersicon esculentum) plants was inhibited by NO donors sodium nitroprusside and S-nitroso-N-acetyl-penicillamine. The inhibition was reversed by supplying the plants with the NO scavenger 2-(4-carboxiphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide. NO also blocked the hydrogen peroxide (H(2)O(2)) production and proteinase inhibitor synthesis that was induced by systemin, oligouronides, and jasmonic acid (JA). However, H(2)O(2) generated by glucose oxidase and glucose was not blocked by NO, nor was H(2)O(2)-induced proteinase inhibitor synthesis. Although the expression of proteinase inhibitor genes in response to JA was inhibited by NO, the expression of wound signaling-associated genes was not. The inhibition of wound-inducible H(2)O(2) generation and proteinase inhibitor gene expression by NO was not due to an increase in salicylic acid, which is known to inhibit the octadecanoid pathway. Instead, NO appears to be interacting directly with the signaling pathway downstream from JA synthesis, upstream of H(2)O(2) synthesis. The results suggest that NO may have a role in down-regulating the expression of wound-inducible defense genes during pathogenesis.     

Proteomic evaluation of wound‐healing processes in potato (Solanum tuberosum L.) tuber tissue

Proteins from potato (Solanum tuberosum L.) tuber slices, related to the wound‐healing process, were separated by 2‐DE and identified by an MS analysis in MS and MS/MS mode. Slicing triggered differentiation processes that lead to changes in metabolism, activation of defence and cell‐wall reinforcement. Proteins related to storage, cell growth and division, cell structure, signal transduction, energy production, disease/defence mechanisms and secondary metabolism were detected. Image analysis of the 2‐DE gels revealed a time‐dependent change in the complexity of the polypeptide patterns. By microscopic observation the polyalyphatic domain of suberin was clearly visible by D4, indicating that a closing layer (primary suberisation) was formed by then. A PCA of the six sampling dates revealed two time phases, D0–D2 and D4–D8, with a border position between D2 and D4. Moreover, a PCA of differentially expressed proteins indicated the existence of a succession of proteomic events leading to wound‐periderm reconstruction. Some late‐expressed proteins (D6–D8), including a suberisation‐associated anionic peroxidase, have also been identified in the native periderm. Despite this, protein patterns of D8 slices and native periderm were still different, suggesting that the processes of wound‐periderm formation are extended in time and not fully equivalent. The information presented in this study gives clues for further work on wound healing‐periderm formation processes.     

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.     

Wound healing and induced resistance in potato tubers

It was demonstrated that biogenic elicitors, arachidonic acid and chitosan, locally and systemically stimulated wound healing in potato tuber tissues by increasing the number of wound periderm layers, accelerating the development of cork cambium (phellogen), and inducing proteinase inhibitors. The signal molecules, jasmonic and salicylic acids, had different effects on the development of wound periderm: jasmonic acid locally and systemically stimulated potato wound healing and elevated the level of proteinase inhibitors, whereas salicylic acid did not have any effect on wound healing and even blocked the formation of proteinase inhibitors.     

水分胁迫诱导玉米叶片质外体产生H2O2机理

通过组织化学染色、电镜观察、酶活性分析对水分胁迫诱导玉米叶片质外体产生H2O2进行了研究。结果表明:水分胁迫能够诱导玉米叶片内源ABA的积累,ABA参与了水分胁迫诱导的玉米叶片H2O2的产生,质膜NADPH氧化酶、细胞壁过氧化物酶(POD)以及质外体多胺氧化酶(PAO)是水分胁迫诱导玉米细胞在质外体产生H2O2的来源,其中质膜NADPH氧化酶是主要来源;内源ABA的积累参与了水分胁迫激活的质膜NADPH氧化酶、细胞壁POD和质外体PAO活性的提高。研究认为,水分胁迫诱导玉米细胞在质外体产生H2O2可能是由于水分胁迫下内源ABA的积累通过激活质膜NADPH氧化酶、细胞壁POD以及质外体PAO的活性而实现的。     

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.     

ABA诱导玉米叶质外体H2O2积累的机制

通过组织化学染色和电镜观察并结合酶活性分析表明,ABA可通过诱导玉米（Zea mays L、）叶片质膜NADPH氧化酶、细胞壁POD及质外体PAO活性的升高,使其质外体产生H2O2;其中质膜NADPH氧化酶起主要作用。     

Copper amine oxidase expression in defense responses to wounding and Ascochyta rabiei invasion

Wounding chickpea (Cicer arietinum) internodes or cotyledons resulted in an increase in the steady-state level of copper amine oxidase (CuAO) expression both locally and systemically. Dissection of the molecular mechanisms controlling CuAO expression indicated that jasmonic acid worked as a potent inducer of the basal and wound-inducible CuAO expression, whereas salicylic acid and abscisic acid caused a strong reduction of the wound-induced CuAO expression, without having any effect on the basal levels. Epicotyl treatment with the CuAO mechanism-based inhibitor 2-bromoethylamine decreased hydrogen peroxide (H(2)O(2)) levels in all the internodes, as evidenced in vivo by 3,3'-diaminobenzidine oxidation. Moreover, inhibitor pretreatment of wounded epicotyls resulted in a lower accumulation of H(2)O(2) both at the wound site and in distal organs. In vivo CuAO inhibition by 2-bromoethylamine after inoculation of resistant chickpea cv Sultano with Ascochyta rabiei resulted in the development of extended necrotic lesions, with extensive cell damage occurring in sclerenchyma and cortical parenchyma tissues. These results, besides stressing the fine-tuning by key signaling molecules in wound-induced CuAO regulation, demonstrate that local and systemic CuAO induction is essential for H(2)O(2) production in response to wounding and indicate the relevance of these enzymes in protection against pathogens.     

系统素、茉莉酸在番茄系统伤反应中的作用

当植物受到机械损伤或昆虫伤害时,植物体会在受伤部位产生伤信号分子启动防御基因的系统表达,蛋白酶抑制剂基因是防御基因的一典型代表.番茄是研究植物系统伤信号很好的模式植物,目前,三种类型的番茄系统伤信号突变体被鉴定出来,通过对番茄系统伤信号突变体进行功能分析并在它们之间进行相互嫁接实验,研究结果表明系统素和茉莉酸通过同一信号通路来激活防御基因的系统表达.系统素(或它的前体原系统素)在受伤部位激活茉莉酸的合成,使之达到系统反应的水平,应对外来伤害;茉莉酸或其衍生物是重要的系统伤信号分子,它诱导伤防御基因的系统表达.植物的系统伤反应可比做动物的炎症反应,它们之间有许多相似之处.     

Induction of polyamine oxidase 1 by Helicobacter pylori causes macrophage apoptosis by hydrogen peroxide release and mitochondrial membrane depolarization

Helicobacter pylori infects the human stomach by escaping the host immune response. One mechanism of bacterial survival and mucosal damage is induction of macrophage apoptosis, which we have reported to be dependent on polyamine synthesis by arginase and ornithine decarboxylase. During metabolic back-conversion, polyamines are oxidized and release H(2)O(2), which can cause apoptosis by mitochondrial membrane depolarization. We hypothesized that this mechanism is induced by H. pylori in macrophages. Polyamine oxidation can occur by acetylation of spermine or spermidine by spermidine/spermine N(1)-acetyltransferase prior to back-conversion by acetylpolyamine oxidase, but recently direct conversion of spermine to spermidine by the human polyamine oxidase h1, also called spermine oxidase, has been demonstrated. H. pylori induced expression and activity of the mouse homologue of this enzyme (polyamine oxidase 1 (PAO1)) by 6 h in parallel with ornithine decarboxylase, consistent with the onset of apoptosis, while spermidine/spermine N(1)-acetyltransferase activity was delayed until 18 h when late stage apoptosis had already peaked. Inhibition of PAO1 by MDL 72527 or by PAO1 small interfering RNA significantly attenuated H. pylori-induced apoptosis. Inhibition of PAO1 also significantly reduced H(2)O(2) generation, mitochondrial membrane depolarization, cytochrome c release, and caspase-3 activation. Overexpression of PAO1 by transient transfection induced macrophage apoptosis. The importance of H(2)O(2) was confirmed by inhibition of apoptosis with catalase. These studies demonstrate a new mechanism for pathogen-induced oxidative stress in macrophages in which activation of PAO1 leads to H(2)O(2) release and apoptosis by a mitochondrial-dependent cell death pathway, contributing to deficiencies in host defense in diseases such as H. pylori infection.     

Systemic signaling in the wound response

In many plants, localized tissue damage elicits an array of systemic defense responses against herbivore attack. Progress in our understanding of the long-distance signaling events that control these responses has been aided by the identification of mutants that fail to mount systemic defenses in response to wounding. Grafting experiments conducted with various mutants of tomato indicate that systemic signaling requires both the biosynthesis of jasmonic acid at the site of wounding and the ability to perceive a jasmonate signal in remote tissues. These and other studies support the hypothesis that jasmonic acid regulates the production of, or acts as, a mobile wound signal. Following its synthesis in peroxisomes, further metabolism of jasmonic acid might enhance its stability, transport, or action in remote tissues. Recent studies in tomato suggest that the peptide signal systemin promotes long-distance defense responses by amplifying jasmonate production in vascular tissues.     

Involvement of hydrogen peroxide and nitric oxide in expression of the ipomoelin gene from sweet potato

The IPO (ipomoelin) gene was isolated from sweet potato (Ipomoea batatas cv Tainung 57) and used as a molecular probe to investigate its regulation by hydrogen peroxide (H(2)O(2)) and nitric oxide (NO) after sweet potato was wounded. The expression of the IPO gene was stimulated by H(2)O(2) whether or not the plant was wounded, but its expression after wounding was totally suppressed by the presence of diphenylene iodonium, an inhibitor of NADPH oxidase, both in the local and systemic leaves of sweet potato. These results imply that a signal transduction resulting from the mechanical wounding of sweet potato may involve NADPH oxidase, which produces endogenous H(2)O(2) to stimulate the expression of the IPO gene. The production of H(2)O(2) was also required for methyl jasmonate to stimulate the IPO gene expression. On the contrary, NO delayed the expression of the IPO gene, whereas N(G)-monomethyl-L-arginine monoacetate, an inhibitor of NO synthase, enhanced the expression of the IPO gene after the plant was wounded. This study also demonstrates that the production of H(2)O(2) stained with 3,3'-diaminobenzidine hydrochloride could be stimulated by wounding but was suppressed in the presence of NO. Meanwhile, the generation of NO was visualized by confocal scanning microscope in the presence of 4,5-diaminofluorescein diacetate after sweet potato was wounded. In conclusion, when sweet potato was wounded, both H(2)O(2) and NO were produced to modulate the plant's defense system. Together, H(2)O(2) and NO regulate the expression of the IPO gene, and their interaction might further stimulate plants to protect themselves from invasions by pathogens and herbivores.     

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