Isozyme profiles associated with the hypersensitive response of Chenopodium foetidum to plum pox virus infection

Isozyme profiles of esterases (E.C. 3.1.1.1), glutamate oxaloacetate transaminase (E.C. 2.6.1.1) and peroxidases (E.C. 1.11.1.7) have been determined in healthy tissues of Chenopodium foetidum as well as their modifications during leaf development. The effect of plum pox virus infection on the isozyme profiles has also been studied. Virus-induced necrotic lesions displayed a peroxidase (POX) pattern that has not been found in any other tissue of the plant so far analyzed. The pattern was similar to that of old yellow leaves, except that POX-B, which was detected in the necrotic lesions, has not been detected in any developmental stage of healthy leaves. Changes in the peroxidase profile seem to begin early during infection, even before necrosis is visible. We suggest that senescence is established at necrotic lesions extending from there to the rest of the infected leaf affecting the peroxidase isozyme pattern. However, other changes, which induce POX-B, must also take place at necrotic lesions. These do not extend to the rest of the infected leaves. Plum pox virus infection has less effect on the glutamate oxaloacetate transaminase and esterase isozyme patterns, inducing an almost normal senescence pattern.     

Comparison among the isozyme profiles associated with ethrel treatments of leaves, and with senescence and plum pox virus infection in Chenopodium foetidum

Changes in the isozyme profiles of peroxidases (POX, EC 1.11.1.7), glutamate oxaloacetate transaminase (GOT, EC 2.6.1.1) and esterases (EST, EC 3.1.1.1) have been studies in leaves of Chenopodium foetidum S. treated with ethrel. Different systems for administration of the ethrel led to different responses. In intact plants, treatment of the complete surface of leaves or local administration by pricking the leaves induced senescence and wilting as well as quick changes, characteristic of ageing, in the isozyme patterns of the treated leaves. Isolated leaves treated with ethrel in vitro also showed a senescence response, but this was followed by a necrosis that displayed an isozyme pattern highly similar to that of necrotic lesions induced by plum pox virus (PPV) infection. An accelerated senescence process seems to be involved in the induction of changes in the isozyme patterns of expression during the hypersensitive response of Chenopodium foetidum to PPV infection, and ethylene could participate in this process. However, other factors may also be required for necrotization.     

Ten rice peroxidases redundantly respond to multiple stresses including infection with rice blast fungus

Class III plant peroxidases are believed to function in diverse physiological processes including disease resistance and wound response, but predicted low substrate specificities and the presence of 70 or more isoforms have made it difficult to define a specific physiological function(s) for each gene. To select pathogen-responsive POX genes, we analyzed the expression profiles of 22 rice POX genes after infection with rice blast fungus. The expression of 10 POX genes among the 22 genes was induced after fungal inoculation in both compatible and incompatible hosts. Seven of the 10 POX genes were expressed at higher levels in the incompatible host than in the compatible host 6-24 h after inoculation by which time no fungus-induced lesions have appeared. Organ-specific expression and stress-induced expression by wounding and treatment with probenazole, an agrichemical against blast fungus, jasmonic acid, salicylic acid and 1-aminocyclopropane-1-carboxylate, a precursor of ethylene, indicated that rice POXs have individual characteristics and can be classified into several types. A comparison of the amino acid sequences of POXs showed that multiple isoforms with a high sequence similarity respond to stress in different or similar ways. Such redundant responses of POX genes may guarantee POX activities that are necessary for self-defense in plant tissues against environmental stresses including pathogen infection.     

Rapid accumulation of trihydroxy oxylipins and resistance to the bean rust pathogen Uromyces fabae following wounding in Vicia faba

BACKGROUND AND AIMS: Insect damage to plants leads to wound-activated responses directed to healing of damaged tissues, as well as activation of defences to prevent further insect damage. Negative cross-talk exists between the jasmonic acid-based signalling system that is activated upon insect attack and the salicylic acid-based system frequently activated following pathogen infection. Thus, insect attack may compromise the ability of the plant to defend itself against pathogens and vice versa. However, insect herbivory and mechanical wounding have been shown to reduce fungal infections on some plants, although the underlying mechanisms remain to be defined. This work examines the effects of mechanical wounding on rust infection both locally and systemically in the broad bean, Vicia faba and follows changes in oxylipins in wounded leaves and unwounded leaves on wounded plants. METHODS: The lamina of first leaves was wounded by crushing with forceps, and first and second leaves were then inoculated, separately, with the rust Uromyces fabae at various times over a 24 h period. Wounded first leaves and unwounded second leaves were harvested at intervals over a 24 h period and used for analysis of oxylipin profiles. KEY RESULTS Mechanical wounding of first leaves of broad bean led to significantly reduced rust infection in the wounded first leaf as well as the unwounded second leaf. Increased resistance to infection was induced in plants inoculated with rust just 1 h after wounding and was accompanied by rapid and significant accumulation of jasmonic acid and two trihydroxy oxylipins in both wounded first leaves and unwounded second leaves. The two trihydroxy oxylipins were found to possess antifungal properties, reducing germination of rust spores. CONCLUSIONS: These results demonstrate the rapidity with which resistance to pathogen infection can be induced following wounding and provides a possible mechanism by which pathogen infection might be halted.     

Trypsin inhibitor activity in mature tobacco and tomato plants is mainly induced locally in response to insect attack,wounding and virus infection

Wounding of plants by insects is often mimicked in the laboratory by mechanical means such as cutting or crushing, and has not been compared directly with other forms of biotic stress such as virus infection. To compare the response of plants to these types of biotic and abiotic stress, trypsin inhibitor (TI) activity induced locally and systemically in mature tobacco (Nicotiana tabacum L.) and tomato (Lycopersicon esculentum L.) plants was followed for 12 days. In tobacco, cutting, crushing and insect feeding all induced comparable levels of TI activity of approx. 5 nmol·(mg leaf protein)^?1 in wounded leaves, while tobacco mosaic virus (TMV) infection of tobacco induced 10-fold lower amounts in the infected leaves. In tomato, feeding by insects also led to the induction of a level of TI activity of 5 nmol·(mg leaf protein)^?1. In contrast, both cutting and crushing of tomato leaves induced 10-fold higher amounts. These data show that biotic stress, in the form of insect feeding and TMV infection, and abiotic stress, in the form of wounding, have different effects on local levels of induced TI activity in mature tobacco and tomato plants. Irrespective of the type of wounding, in neither tobacco nor tomato could systemic induction of TI activity be observed in nearby unwounded leaves, which suggests that systemic induction of TI activity in mature tobacco and tomato plants is different from systemic TI induction in seedlings. Wounding of tobacco leaves, however, did increase the responsiveness to wounding elsewhere in the plant, as measured by an increased induction of TI activity.     

Wound-induced expression of a tobacco peroxidase is not enhanced by ethephon and suppressed by methyl jasmonate and coronatine

In tobacco plants, wounding induces production of a set of defense-related proteins such as basic pathogenesis-related (PR) proteins and proteinase inhibitors (PIs) via the jasmonate/ethylene pathway. Although class III plant peroxidase (POX) is also wound-inducible, the regulatory mechanism for its wound-induced expression is not fully understood. Here, we describe that a tobacco POX gene (tpoxN1), which is constitutively expressed in roots, is induced locally 30 min after wounding and then systemically in tobacco plants. Infection of necrotizing virus also induced tpoxN1 gene. The wound-induced expression was not enhanced by known wound-signal compounds such as methyl jasmonate (MeJA) and ethephon in contrast to other wound-inducible genes such as basic PR-1 and PI-II genes. And treatment with MeJA and coronatine, biological analogs of jasmonate, rather suppressed the tpoxN1 expression. Salicylic acid, an antagonist of jasmonate-based wound signaling, did not suppress the wound-induced expression of tpoxN1. Only spermine, which is reported as an endogenous inducer for acidic PR genes in tobacco mosaic virus-infected tobacco leaves, could induce tpoxN1 gene expression. These results suggest that wound-induced expression of the tpoxN1 gene is regulated differently from that of the basic PR and PI-II genes.     

Effect of wounding on ethylene biosynthesis and senescence of detached spinach leaves

Parameters of senescence and ethylene biosynthesis pathway were screened simultaneously in detached spinach leaves and leaf discs. Senescence was enhanced by application of 1-aminocyclopropane-1-carboxylic acid (ACC) and was retarded by amino-ethoxyvinylglycine (AVG). Evidence is presented showing that the bursts of both wound- and climacteric-like ethylene promoted senescence of detached leaves and leaf discs. This ethylene-enhanced leaf senescence was dependent on: (a) ethylene production rates in the tissue; (b) the degree of wounding. Wounding resulted in elevated levels of 1-(malonylamino)cyclopropane-1-carboxylic acid (MACC), which declined in advanced stages of senescence. The results suggest that wounding might be regarded as one of the primary events in the induction of the senescence syndrome in detached leaves and leaf discs, while ethylene is implicated as a regulator of the rate of the process.     

Kinetics of the cell biological changes occurring in the progression of DNA damage-induced senescence

Cellular senescence is characterized by cell-cycle arrest accompanied by various cell biological changes. Although these changes have been heavily relied on as senescence markers in numerous studies on senescence and its intervention, their underlying mechanisms and relationship to each other are poorly understood. Furthermore, the depth and the reversibility of those changes have not been addressed previously. Using flow cytometry coupled with confocal microscopy and Western blotting, we quantified various senescence-associated cellular changes and determined their time course profiles in MCF-7 cells undergoing DNA damage-induced senescence. The examined properties changed with several different kinetics patterns. Autofluorescence, side scattering, and the mitochondria content increased progressively and linearly. Cell volume, lysosome content, and reactive oxygen species (ROS) level increased abruptly at an early stage. Meanwhile, senescence associated β-galactosidase activity increased after a lag of a few days. In addition, during the senescence progression, lysosomes exhibited a loss of integrity, which may have been associated with the accumulation of ROS. The finding that various senescence phenotypes matured at different rates with different lag times suggests multiple independent mechanisms controlling the expression of senescence phenotypes. This type of kinetics study would promote the understanding of how cells become fully senescent and facilitate the screening of methods that intervene in cellular senescence.     

Paclobutrazol delayed dark-induced senescence of mung bean leaves

Sixty days old mung beans Vigna radiata (L.) Wilczek were treated with soil applied paclobutrazol, at the rate of 500 μg per 10 inch pot. After seven days of application, the plants along with untreated controls were transferred to the dark for induction of senescence. The treated plants exhibited higher chlorophyll content and activity of catalase (CAT) compared to controls. In contrast, control leaves had higher activity of peroxidase (POX) and a higher content of malondialdehyde (MDA), while superoxide dismutase (SOD) activity remained unchanged. Upon transfer to dark, chlorophyll content declined in both control and treated plants but the decline was much faster in control. The activity of CAT decreased significantly in controls while POX activity and MDA content remained higher in control than in treated plants. Paclobutrazol delayed the dark-induced senescence in attached mung bean leaves in association with the maintenance of higher activity of CAT, low activity of POX, and low MDA contents. The variation in SOD activity was not discernible with senescence levels.     

The apoplastic antioxidant system in Prunus: response to long-term plum pox virus infection

This work describes, for the first time, the changes taking place in the antioxidative system of the leaf apoplast in response to plum pox virus (PPV) in different Prunus species showing different susceptibilities to PPV. The presence of p-hydroxymercuribenzoic acid (pHMB)-sensitive ascorbate peroxidase (APX) (class I APX) and pHMB-insensitive APX (class III APX), superoxide dismutase (SOD), peroxidase (POX), NADH-POX, and polyphenoloxidase (PPO) was described in the apoplast from both peach and apricot leaves. PPV infection produced different changes in the antioxidant system of the leaf apoplast from the Prunus species, depending on their susceptibility to the virus. In leaves of the very susceptible peach cultivar GF305, PPV brought about an increase in class I APX, POX, NADH-POX, and PPO activities. In the susceptible apricot cultivar Real Fino, PPV infection produced a decrease in apoplastic POX and SOD activities, whereas a strong increase in PPO was observed. However, in the resistant apricot cultivar Stark Early Orange, a rise in class I APX as well as a strong increase in POX and SOD activities was noticed in the apoplastic compartment. Long-term PPV infection produced an oxidative stress in the apoplastic space from apricot and peach plants, as observed by the increase in H2O2 contents in this compartment. However, this increase was much higher in the PPV-susceptible plants than in the resistant apricot cultivar. Only in the PPV-susceptible apricot and peach plants was the increase in apoplastic H2O2 levels accompanied by an increase in electrolyte leakage. No changes in the electrolyte leakage were observed in the PPV-inoculated resistant apricot leaves, although a 42% increase in the apoplastic H2O2 levels was produced. Two-dimensional electrophoresis analyses revealed that the majority of the polypeptides in the apoplastic fluid had isoelectric points in the range of pI 4-6. The identification of proteins using MALDI-TOF (matrix-assisted laser desorption/ionization-time of flight) and peptide mass fingerprinting analyses showed the induction of a thaumatin-like protein as well as the decrease of mandelonitrile lyase in peach apoplast due to PPV infection. However, most of the selected polypeptides showed no homology with known proteins. This fact emphasizes that, at least in Prunus, most of the functions of the apoplastic space remain unknown. It is concluded that long-term PPV infection produced an oxidative stress in the leaf apoplast, contributing to the deleterious effects produced by PPV infection in leaves of inoculated, susceptible Prunus plants.     

Tissue specificity of tobacco peroxidase isozymes and their induction by wounding and tobacco mosaic virus infection

Peroxidases (EC 1.11.1.7) have been implicated in the responses of plants to physical stress and to pathogens, as well as in a variety of cellular processes including cell wall biosynthesis. Tissue samples from leaf, root, pith, and callus of Nicotiana tabacum were assayed for specific peroxidase isozymes by analytical isoelectric focusing. Each tissue type was found to exhibit a unique isozyme fingerprint. Root tissue expressed all of the detectable peroxidase isozymes in the tobacco plant, whereas each of the other tissues examined expressed a different subset of these isozymes. In an effort to determine which peroxidase isozymes from Nicotiana tabacum are involved in cell wall biosynthesis or other normal cellular functions and which respond to stress, plants were subjected to either wounding or infection with tobacco mosaic virus. Wounding the plant triggered the expression of several cationic isozymes in the leaf and both cationic and anionic isozymes in pith tissue. Maximum enzyme activity was detected at 72 hours after wounding, and cycloheximide treatment prevented this induction. Infection of tobacco with tobacco mosaic virus induced two moderately anionic isozymes in the leaves in which virus was applied and also systemically induced in leaves which were not inoculated with virus.     

Changes in the antioxidative metabolism induced by Apple chlorotic leaf spot virus infection in peach [Prunus persica (L.) Batsch]

Apple chlorotic leaf spot virus (ACLSV) is the causal agent of “viruela” disease, one of the limiting factors to apricot production in affected areas in the Southeast of Spain. In this work, the response of antioxidant enzymes to ACLSV infection of an indicator peach genotype, ‘GF305’, which is characterised by a great susceptibility to this virus, was studied before (short-period incubation) and after (long-period incubation) a cold treatment. Short-period ACLSV incubation caused significant changes in ascorbate peroxidase (APX), peroxidase (POX) and catalase (CAT) activities. In addition, long-period ACLSV incubation caused significant changes of activities in most of the antioxidant enzymes examined. The results show increases in the APX, dehydroascorbate reductase (DHAR), superoxide dismutase (SOD) and glutathione-S-transferase (GST) activities, whereas POX suffered a decrease of about 34%.No changes in lipid peroxidation, measured as TBARS, were observed in peach leaves as a consequence of the long-period ACLSV incubation. Overall, the data show that long-period ACLSV incubation did not produce any symptoms in peach GF305 leaves or damage to membranes (no changes in lipid peroxidation), and this response was correlated with an increase in the antioxidant defences in leaves, such as the ASC-GSH cycle enzymes and the SOD and GST activities.     

Effect of phytoplasma infection on metabolite content and antioxidant enzyme activity in lime (<Emphasis Type="Italic">Citrus aurantifolia</Emphasis>)

The objective of the present work was to study biochemical alterations in lime plants infected by the Candidatus Phytoplasma aurantifoliae. Changes in antioxidant activities, content of chlorophylls (Chl), carotenoids (Car), soluble proteins, sugars and auxin (IAA) in infected plant were investigated. The activities of polyphenol oxidase (PPO), peroxidase (POX) and superoxide dismutase (SOD) were observed to be greater in infected leaves than the healthy control. Also according to non-denaturing PAGE, in infected leaves all the antioxidative enzymes isoforms were stronger than that of the healthy control. These results suggest that antioxidant enzymes can be activated in response to infection by phytoplasma. The decrease in content of proteins, total soluble and reducing sugars in infected plants point out changes in host metabolism due to the phytoplasma infection. The reduction in chlorophylls and auxin content shows that the phytoplasma can interfere in photosynthesis and induces senescence in the leaf. In conclusion, this study provides new insights into the lime response to phytoplasma infection.