Undamaged Tissue Research Articles

Nuclear export of the nonenveloped parvovirus virion is directed by an unordered protein signal exposed on the capsid surface.

It is uncertain whether nonenveloped karyophilic virus particles may actively traffic from the nucleus outward. The unordered amino-terminal domain of the VP2 major structural protein (2Nt) of the icosahedral parvovirus minute virus of mice (MVM) is internal in empty capsids, but it is exposed outside of the shell through the fivefold axis of symmetry in virions with an encapsidated single-stranded DNA genome, as well as in empty capsids subjected to a heat-induced structural transition. In productive infections of transformed and normal fibroblasts, mature MVM virions were found to efficiently exit from the nucleus prior to cell lysis, in contrast to the extended nuclear accumulation of empty capsids. Newly formed mutant viruses lacking the three phosphorylated serine residues of 2Nt were hampered in their exit from the human transformed NB324K nucleus, in correspondence with the capacity of 2Nt to drive microinjected phosphorylated heated capsids out of the nucleus. However, in normal mouse A9 fibroblasts, in which the MVM capsid was phosphorylated at similar sites but with a much lower rate, the nuclear exit of virions and microinjected capsids harboring exposed 2Nt required the infection process and was highly sensitive to inhibition of the exportin CRM1 in the absence of a demonstrable interaction. Thus, the MVM virion exits the nucleus by accessing nonconventional export pathways relying on cell physiology that can be intensified by infection but in which the exposure of 2Nt remains essential for transport. The flexible 2Nt nuclear transport signal may illustrate a common structural solution used by nonenveloped spherical viruses to propagate in undamaged host tissues.

Damaging effect of the fish pathogen Aeromonas salmonicida ssp. salmonicida on intestinal enterocytes of Atlantic salmon (Salmo salar L.)

In fish, bacterial pathogens can enter the host by one or more of three different routes: (a) skin, (b) gills and (c) gastrointestinal tract. Bacteria can cross the gastrointestinal lining in three different ways. In undamaged tissue, bacteria can translocate by transcellular or paracellular routes. Alternatively, bacteria can damage the intestinal lining with extracellular enzymes or toxins before entering. Using an in vitro (Ussing chamber) model, this paper describes intestinal cell damage in Atlantic salmon (Salmo salar L.) caused by the fish pathogen Aeromonas salmonicida ssp. salmonicida, the causative agent of furunculosis. The in vitro method clearly demonstrated substantial detachment of enterocytes from anterior region of the intestine (foregut) upon exposure to the pathogen. In the hindgut (posterior part of the intestine), little detachment was observed but cellular damage involved microvilli, desmosomes and tight junctions. Based on these findings, we suggest that A. salmonicida may obtain entry to the fish by seriously damaging the intestinal lining. Translocation of bacteria through the foregut (rather than the hindgut) is a more likely infection route for A. salmonicida infections in Atlantic salmon.

Central sensitization and cutaneous hyperalgesia

Injury to the skin results in exaggerated pain both at the site of injury as well as in surrounding undamaged tissues. In this study the general organization of the somatosensory system is briefly reviewed, and then the neural mechanisms that account for hypersensitivity after cutaneous injury are discussed. In brief, exaggerated responsiveness, or sensitization, of primary afferent fibers in the skin account for hyperalgesia at the site of injury, whereas sensitization of spinal projection neurons, in particular spinothalamic and spinomesencephalic neurons, account for hyperalgesia in undamaged tissues surrounding the injury site.

Fatal trauma to the neck: immunohistochemical study of local injuries

As fatal trauma to the neck is often associated with short survival times, proof of vitality may often be difficult using standard histochemical techniques. Soft tissue neck injuries resulting from strangulation by ligature or manual strangulation were examined immunohistochemically using antibodies to myoglobin, fibronectin, C5b-9 and MRP14, and compared to controls consisting of accidental soft tissue neck injuries as well as undamaged neck soft tissue. Although survival times in the study and control groups were unknown and certainly some individual variation may be expected in the time course of normal wound development, both the study and control groups demonstrated similar time courses in the immunohistochemical detection of antigen. Myoglobin was always found in those samples in which only one antigen was shown to be involved in an injury-specific pattern; myoglobin and fibronectin were found in samples with dual antigen involvement. Samples involving three antigens always included C5b-9 in addition to myoglobin and fibronectin. The single positive MRP14 sample in the study and control groups was simultaneously positive for the other markers used. Myoglobin, fibronectin, C5b-9 and MRP14 are therefore suitable for immunohistochemical detection of vital reactions and estimation of temporal relationships in the early posttraumatic period after neck trauma.

Activation of ERK1/2 after neonatal rat cerebral hypoxia-ischaemia.

Activation of extracellular signal-related kinases (ERK1/2), also known as p42/44 mitogen-activated protein kinase (MAPK), is considered important for neuronal survival, cell proliferation and apoptosis. In the present study, activation (phosphorylation) of ERK1/2 (P-ERK) was investigated in brains of 7-day-oldrats after hypoxia-ischaemia (HI). In damaged areas, P-ERK-positive neurons appeared immediately after HI and the staining remained for at least 8 h. At later time points, 24 and 72 h post-HI, P-ERK-positive neurons were found in the core of the infarct and in the border zone to undamaged tissue. These cells also showed signs of DNA damage and calpain-induced fodrin breakdown, indicative of injury. At 72 h post-HI, P-ERK was also observed in microglia in the border zone to the damaged area and in astrocytes and oligodendrocytes in white matter of both hemispheres. P-ERK was strongly expressed in the subventricular zone in both hemispheres after HI at most time points, although the staining in the ipsilateral (damaged hemisphere) was stronger than in the contralateral (non-damaged hemisphere). In summary, ERK1/2 activation occurred early in neurons after HI in the neonatal brain, and mainly in cells displaying signs of damage.

Gene treatment of cerebral stroke by rAAV vector delivering IL-1ra in a rat model.

In this study, we injected recombinant adeno-associated virus (rAAV) vectors expressing the interleukin-1 receptor antagonist (rAAV-IL-1ra) into the cortex of rats experiencing transient cerebral ischemia. An accumulation of IL-1ra in cortical tissues of rAAV-IL-1ra-injected animals was confirmed by ELISA. Triphenyltetrazolium chloride (TTC) staining of viable brain tissue revealed that the rAAV-delivered IL-1ra gene could rescue the brain tissues from ischemia-induced injury. Cortical tissues that received rAAV-IL-1ra injections had both significantly smaller total volumes of infarction as well as smaller areas of infarction on each brain slice when compared with the control models. In situ labeling analysis demonstrated significant reduction of apoptotic cells in cortical tissues rescued by rAAV-IL-1ra. Immunohistochemistry staining revealed that the rescued brain tissues contained the same levels of neuronal cells as contralateral undamaged brain tissues. These findings confirmed that the rAAV delivering the IL-1ra gene is a potential therapy for stroke.

The Effects of L-Carnitine L-Tartrate Supplementation on Hormonal Responses to Resistance Exercise and Recovery

The purpose of this investigation was to examine the influence of L-carnitine L-tartrate (LCLT) supplementation using a balanced, cross-over, placebo-controlled research design on the anabolic hormone response (i.e., testosterone [T], insulin-like growth factor-I, insulin-like growth factor-binding protein-3 [IGFBP-3], and immunofunctional and immunoreactive growth hormone [GHif and GHir]) to acute resistance exercise. Ten healthy, recreationally weight-trained men (mean +/- SD age 23.7 +/- 2.3 years, weight 78.7 +/- 8.5 kg, and height 179.2 +/- 4.6 cm) volunteered and were matched, and after 3 weeks of supplementation (2 g LCLT per day), fasting morning blood samples were obtained on six consecutive days (D1-D6). Subjects performed a squat protocol (5 sets of 15-20 repetitions) on D2. During the squat protocol, blood samples were obtained before exercise and 0, 15, 30, 120, and 180 minutes postexercise. After a 1-week washout period, subjects consumed the other supplement for a 3-week period, and the same experimental protocol was repeated using the exact same procedures. Expected exercise-induced increases in all of the hormones were observed for GHir, GHif, IGFBP-3, and T. Over the recovery period, LCLT reduced the amount of exercise-induced muscle tissue damage, which was assessed via magnetic resonance imaging scans of the thigh. LCLT supplementation significantly (p < 0.05) increased IGFBP-3 concentrations prior to and at 30, 120, and 180 minutes after acute exercise. No other direct effects of LCLT supplementation were observed on the absolute concentrations of the hormones examined, but with more undamaged tissue, a greater number of intact receptors would be available for hormonal interactions. These data support the use of LCLT as a recovery supplement for hypoxic exercise and lend further insights into the hormonal mechanisms that may help to mediate quicker recovery.

Using array hybridization to monitor gene expression at the single cell level.

Advances in high-throughput genome sequencing demand the development of more efficient ways of examining gene expression at a cellular level. During recent years, polymerase chain reaction (PCR)-based methods have been developed that allow the amplification of mRNA from small amounts of material, even from single animal cells. In parallel, several analytical tools permit a global monitoring of gene expression. To date, high throughput analysis methods have not been accessible for single plant cell samples. In the protocol described here, cDNA array hybridization (expression profiling) and an amplification strategy using reverse transcriptase PCR are merged with high spatial resolution sampling from undamaged plant tissue. This protocol gives us a new tool to examine tissue-specific gene expression patterns on a comprehensive scale. To demonstrate the usefulness of this tool, gene expression patterns in samples from Arabidopsis thaliana L. cv. C24 leaf epidermal and mesophyll cells were measured; several differentially expressed genes were identified when single cell samples were compared. The protocol described has the potential of increasing the efficiency of tissue-specific expression analysis by combining high-throughput profiling with straightforward sampling and amplification procedures.

Ozone-response mechanisms in tobacco: implications of polyamine metabolism.

• Polyamines have been suggested to counteract oxidative damage in plants. Here, we present a detailed analysis of polyamine accumulation and its relationship to photosynthetic parameters in two tobacco (Nicotiana tabacum) cultivars (ozone-sensitive Bel W3 and ozone-tolerant Bel B) after a single ozone pulse and after a 1-month exposure in the open air. • Free putrescine accumulated in undamaged tissue of both cultivars, whereas putrescine conjugated to soluble and cell-wall bound components accumulated predominantly in tissue undergoing cell death in Bel W3 plants. Accumulation was caused by a redirection of the conjugation pathway, as well as by a transient increase in arginine decarboxylase and ornithine decarboxylase specific activity. This increase seemed to be regulated at post-transcriptional level. • Measurements of chlorophyll content and fluorescence showed that, in addition to visible necrotic lesions, Bel W3 plants suffered considerable photosynthetic damage in other parts of the leaf. • Accumulation of conjugated putrescine is part of the ozone-induced programmed cell death response in Bel W3 plants. Ozone-induced synthesis of free putrescine is not correlated with ozone-resistance in Bel B plants, which are apparently impaired in signal transduction pathways that are necessary to control the cellular redox state. However, Bel B plants are able to perceive ozone stress and to induce a series of defense mechanisms without activating hypersensitive cell death.

Persistent secondary hyperalgesia after gastrocnemius incision in the rat

Secondary hyperalgesia, an exaggerated response to stimuli applied to undamaged tissue surrounding an injury, is a common consequence of tissue injury and inflammation. It is well established that the etiology of secondary hyperalgesia is sensitization of central neurons but the exact mechanism and its role in certain clinical pain states is unclear. In the present experiments, we studied responses to punctate and non-punctate mechanical stimuli and to heat applied to the plantar aspect of the hindpaw remote to an incision in the gastrocnemius region of the rat hindlimb. Median withdrawal thresholds to von Frey filaments were reduced 2h after incision of skin, fascia and muscle (gastrocnemius incision, n = 9) and remained reduced through postoperative day 6 (p < 0.05 vs sham). Only a transient reduction in withdrawal threshold occurred after incision of skin and fascia (skin incision, n = 10). No enhanced responsiveness to blunt mechanical stimulation or reduction in withdrawal latency to heat was present after gastrocnemius incision (p > 0.05vs sham, n = 9 each group). Reduced withdrawal thresholds were blocked by i.t. administration of morphine and by local anesthetic injection at the test site 2h and 2 days after gastrocnemius incision. These pharmacological data provide evidence that reduced withdrawal thresholds after gastrocnemius incision are nociceptive behaviors indicating persistent secondary hyperalgesia. Because the behaviors have a similar time course to secondary hyperalgesia in postoperative patients, the model will be useful to evaluate the mechanisms for secondary mechanical hyperalgesia after incision, its pharmacological characteristics and its potential role in persistent postoperative pain. Copyright 2002 European Federation of Chapters of the International Association for the Study of Pain. Published by Elsevier Science Ltd. All rights reserved.

Laser induced thermotherapy (LITT) of experimental liver metastasis-detection of residual tumors using Gd-DTPA enhanced MRI.

The aim of this study was to evaluate the accuracy of Gd-DTPA MRI in the detection of recurrent tumor after laserinduced thermotherapy (LITT) of experimental liver metastases. LITT was performed at different energy levels in VX-2 tumor-bearing rabbits (n = 80). MRI and histology were placed at 0, 24, 96 hours, and 14 days. Signal intensities were calculated of the transition between thermally damaged and undamaged tissue (transition zone = TZ) and of the surrounding tissue (reference zone = RZ). Tumor recurrence was seen in 47 animals. At 24 hours sensitivity, specificity and accuracy was 92, 100, and 95% in TZ and 23, 100, and 50% in RZ. At 14 days sensitivity, specificity and accuracy was 100, 11, and 60% in TZ and 100, 89, and 95% in RZ. Recurrence is best excluded in TZ at 24 hour and in RZ at 14 day with an accuracy up to 95%.

Sox18 Is Transiently Expressed during Angiogenesis in Granulation Tissue of Skin Wounds with an Identical Expression Pattern to Flk-1 mRNA

Sox18 encodes a member of the Sry-related high mobility group box (SOX) family of developmental transcription factors. Examination of Sox18 expression during embryogenesis has shown that Sox18 is expressed transiently in endothelial cells of developing blood vessels, and mutations in Sox18 have been found to underlie the mouse vascular and hair follicle mutant ragged. In this study we have examined the expression of Sox18 in angiogenesis during wound healing. Full-thickness skin wounds were created in mice, and subsequent expression of vascular endothelial growth factor (VEGF), the VEGF receptor Flk-1, α1 (iv) collagen (Col4a1), and Sox18 were studied using in situ hybridization. As has been previously reported, VEGF was expressed predominantly in the keratinocytes at the wound margins. Sox18 expression was found five days after wounding during capillary sprouting in granulation tissue and persisted through the proliferative phase of healing, but was not detected in fully epithelialized wounds 21 days after wounding. Sox18 mRNA expression was detected in capillaries within the granulation tissue and showed an identical pattern of distribution to Flk-1 and Col4a1 mRNA expression in endothelial cells. Immunostaining with a polyclonal anti-Sox18 antibody showed SOX18 protein localized in capillary endothelial cells within the granulation tissue. Capillaries in the subcutaneous tissue of unwounded skin showed no Sox18 expression. Sox18 may therefore represent a transcription factor involved in the induction of angiogenesis during wound healing and tissue repair, but not in the maintenance of endothelial cells in undamaged tissue.

Potential of Cactoblastis cactorum as a Vector for Fungi Pathogenic to Pricklypear, Opuntia inermis

In Australia, fungi associated with larvae of the biological control agent Cactoblastis cactorum may contribute to the control of the exotic weed pricklypear (Opuntia inermis). C. cactorum larvae were assessed for their ability to vector pathogenic fungi into O. inermis by the infestation of larvae with fungal suspensions. Six fungal isolates caused disease after being carried into the host on external surfaces of larvae, and propagules of one isolate (UQ5109) initiated disease after being transferred from the cladode epidermis into the host by larvae feeding on the plant. Scanning electron microscopy revealed extensive hyphal growth on the external surfaces of larvae infested with several of the isolates. Fungi isolated from field-grown O. inermis cladodes were tested for pathogenicity to this plant in an in vivo plant assay. In total, 152 isolates were screened, 22 of which infected the host in pathogenicity tests. Only 1 (UQ5115) infected undamaged host tissue, whereas the remainder required the host to be wounded before infection could proceed. The majority of isolates were only weakly pathogenic, even when inoculated via wounds, suggesting that most were either saprophytes or weak parasites. This study demonstrates that it is possible for larvae of C. cactorum to transmit fungal pathogens into O. inermis tissue and it has provided a sound basis for future field work to determine the contribution that fungi make to the control of O. inermis.

Increased rate of hemorrhage in SOD2-deficient mice following transient focal cerebral ischemia

7 The development of knock-out (KO) mice with targeted disruption of the inducible manganese superoxide dismutase (SOD2) provided a model for investigating the effects of free radicals by perturbing the enzymatic machinery responsible for their metabolism. Previous research in our laboratory showed that heterozygous SOD2-KO mutants exhibited a 50% decrease in SOD2 activity. These animals were also more susceptible to ischemic damage than their wild-type (WT) counterparts and had a 30% mortality at 24 h following 1 h of focal ischemia. Mortality among SOD2-KO mice was increased when the reperfusion period was extended to 72 h. Therefore, animals in this study (SOD2-KO n=7; WT n=7) were subjected to 30 min of middle cerebral artery occlusion (MCAO), using the intraluminal suture method, which was sufficient to cause extensive and reproducible infarcts at 3 days post-insult as measured from H&amp;E stained sections. Our results showed that 7 out of 7 SOD2-KO animals had hemorrhagic transformations (HT) at 72 h post-MCAO, while only 3 out of 7 WT animals exhibited HT (p=0.02 by Mann-Whitney U-test). Interestingly, the rate of HT did not always correlate with the size or anatomical distribution of the lesion. Hemorrhagic transformations were observed in the ischemic core and ischemic penumbra of both WT and SOD2-KO animals, however the latter also showed HT in histologically undamaged tissue. Three additional animals per group were also injected with Evan’s blue at 64 h post-insult and were perfusion-fixed at 72 h. Fifty μm coronal sections examined under fluorescence showed that SOD2-KO animals had extensive extravasation of the dye compared with their WT counterparts. Previous studies by our group did not show a significant increase in HT in SOD2-KO animals at 24 h post-insult compared with WT controls. This suggests that SOD2-KO animals undergo delayed disruption of the blood-brain barrier integrity and may serve as an ideal model for studying the effects of free radical mediated injury to the vascular endothelium.

Recombinant Adeno-Associated Virus Vector Expressing Glial Cell Line-Derived Neurotrophic Factor Reduces Ischemia-Induced Damage

To explore the potential of using the recombinant adeno-associated viral (rAAV) vector, expressing glial cell line-derived neurotrophic factor (GDNF) as the gene therapy for stroke, we injected rAAV vectors expressing GDNF (rAAV-GDNF) into the cortex of rats which had been experiencing transient bilateral common carotid artery ligation and right middle cerebral artery ligation for 90 min. GDNF levels in cortical tissues of rAAV-GDNF-injected animals were significantly higher than in the control animals injected with rAAV-expressing lacZ (rAAV-lacZ), indicating that rAAV can deliver and express the GDNF gene in cortical tissues. Triphenyltetrazolium chloride tissue stain analysis revealed that the rAAV-delivered GDNF gene could rescue the brain tissues from ischemia-induced injury. Cortical tissues which received rAAV-GDNF injections had both significantly smaller total volumes of infarction and smaller areas of infarction on each brain slice than those which were injected with rAAV-lacZ. An in situ labeling analysis demonstrated significantly less apoptotic cells in cortical tissues rescued by rAAV-GDNF, indicating prevention of apoptosis as the mechanism of cortical cell protection. Moreover, immunohistochemistry staining of Neu-N indicated that the rescued brain tissues contained the same number of Neu-N-positive neuronal cells as contralateral undamaged brain tissues. This provides strong evidence that cortical neuronal cells can be rescued by GDNF gene therapy. Indeed, these findings show that the rAAV is a potential delivery vector of GDNF gene for the therapy of stroke.

Raman spectroscopic imaging markers for fatigue-related microdamage in bovine bone.

Raman spectroscopic markers have been determined for fatigue-related microdamage in bovine bone. Microdamage was induced using a cyclic fatigue loading regime. After loading, the specimens were stained en-bloc with basic fuchsin to facilitate damage visualization and differentiate fatigue-induced damage from cracks generated during subsequent histological sectioning. Bone tissue specimens were examined by light microscopy and hyperspectral near-infrared Raman imaging microscopy. Three regions were defined-tissue with no visible damage, tissue with microcracks, and tissue with diffuse damage. Raman transects, lines of 150-200 Raman spectra, were used for initial tissue surveys. Exploratory factor analysis of the transect Raman spectra has identified spectroscopically distinct chemical microstructures of the bone specimens that correlate with damage. In selected regions of damage, full hyperspectral Raman images were obtained with 1.4-microm spatial resolution. In regions of undamaged tissue, the phosphate nu1 band is found at 957 cm(-1), as expected for the carbonated hydroxyapatic bone mineral. However, in regions of visible microdamage, an additional phosphate nu1 band is observed at 963 cm(-1) and interpreted as a more stoichiometric, less carbonated mineral species. Raman imaging confirms the qualitative relationship between the Raman spectral signature of bone mineral and the type of microdamage in bovine bone. Two tentative explanations for the presence of less carbonated phosphate in damaged regions are proposed.

The Abundance of Heterotrophic Protists Associated with Intertidal Seaweeds

Seaweeds have long been known to support abundant populations of bacteria so it is surprising that few studies have considered the abundance of heterotrophic protists on seaweed surfaces. A year-long study was conducted to investigate the numbers of heterotrophic protists on undamaged and damaged tissue of a number of intertidal species of seaweed. Generally, amoebae and flagellates numbered less than 20 cells cm−2on undamaged seaweed tissue but were found in greater numbers (more than 20cm−2) on damaged tissue. Ciliates were around 1 or 2 cells cm−2on undamaged seaweeds and between 1 and 5cm−2on damaged tissue. Numbers of heterotrophic dinoflagellates and heterotrophic diatoms were similar to those of ciliates in late summer and autumn when seaweeds were producing increased amounts of dissolved organic carbon. By assuming that ‘true’ surface protists inhabited the thin surface film of water on seaweeds, comparisons were drawn with the numbers of protists in nearby open waters. After using appropriate conversion factors, both amoebae and flagellates were more abundant in the surface film (per ml) than in the open water. In particular, surface amoebae averaged around 1800 cells ml−1but only reached around 19 cells ml−1in open water. The results are discussed in relation to the various sources of nutrition available to heterotrophic protists on macroalgae. The high numbers of amoebae suggest that they are the major micropredators of bacteria on seaweed surfaces.

Growth of Escherichia coli O157:H7 in bruised apple (Malus domestica) tissue as influenced by cultivar, date of harvest, and source.

Four of five apple cultivars (Golden Delicious, Red Delicious, McIntosh, Macoun, and Melrose) inoculated with Escherichia coli O157:H7 promoted growth of the bacterium in bruised tissue independent of the date of harvest (i.e., degree of apple ripening) or the source of the apple (i.e., tree-picked or dropped fruit). Apple harvest for this study began 4 September 1998 and ended 9 October, with weekly sampling. Throughout this study, freshly picked (<2 days after harvest) McIntosh apples usually prevented the growth of E. coli O157:H7 for 2 days. Growth of E. coli O157:H7 did occur following 6 days of incubation in bruised McIntosh apple tissue. However, the maximum total cell number was approximately 80-fold less than the maximum total cell number recovered from Red Delicious apples. When fruit was stored for 1 month at 4 degrees C prior to inoculation with E. coli O157:H7, all five cultivars supported growth of the bacterium. For each apple cultivar, the pH of bruised tissue was significantly higher and degrees Brix was significantly lower than the pH and degrees Brix of undamaged tissue regardless of the source. In freshly picked apples, changes in the pH did not occur over the harvest season. Bruised Golden Delicious, McIntosh, and Melrose apple tissue pHs were not significantly different (tree-picked or dropped), and the degrees Brix values of McIntosh, Macoun, and Melrose apple tissue were not significantly different. Single-cultivar preparations of cider did not support growth of E. coli, and the cell concentration of inoculated cider declined over an 11-day test period. The rate of decline in E. coli cell concentration in the McIntosh cider was greater than those in the other ciders tested. The findings of this study suggested that the presence of some factor besides, or in addition to, pH inhibited E. coli growth in McIntosh apples.

Technical Advance: A rapid method for detection of plant gene transcripts from single epidermal, mesophyll and companion cells of intact leaves.

A protocol for the detection of gene transcripts from single plant cells in living, undamaged plant tissue is described. Samples of leaf epidermal, mesophyll and companion cells were extracted by using glass microcapillaries and directly subjected to RT-PCR without any purification steps nor time consuming construction of cDNA libraries. The procedure is not restricted to surface cells or outer cell layers. Even cells from the central region of leaves could be harvested. For identification, companion cells were labelled by expression of the green fluorescent protein under control of a companion cell specific promoter. The described method is applicable to a wide range of plants and genes with different expression levels.

Overexpression of a cytoplasm-localized allene oxide synthase promotes the wound-induced accumulation of jasmonic acid in transgenic tobacco.

Jasmonic acid (JA) is involved in regulating the expression of certain plant defense genes and response to various stresses. JA biosynthesis is hypothesized to occur both in chloroplasts and the cytoplasm. In order to test whether or not a cytosol-localized allene oxide synthase (AOS) can promote JA biosynthesis, transgenic tobacco plants containing a flax AOS cDNA without a chloroplast transit sequence under the control of a tetracycline-inducible promoter were generated. Induction of the flax AOS gene in transgenic plants with chlor-tetracycline (Tc) led to the expression of the flax AOS mRNA and protein, which resulted in high level of metabolism of 13(S)-hydroperoxyoctadecatrienoic acid (13(S)-HPOT) and formation of 12-oxo-phytodienoic acid (12-O-PDA). Subcellular fractionation demonstrated that the flax AOS protein and activity were associated with the cytosol. Overexpression of the flax AOS in induced transgenic plants did not increase JA levels in healthy, undamaged leaf tissues. However, in wounded tissues overexpressing a flax AOS, levels of JA and the transcript of a pathogenesis-related gene (PR-1) dramatically increased when compared to those not expressing the flax AOS. Analysis of the release of wound-induced C6 volatiles showed that the level of (Z)-3-hexen-1-ol decreased about 30% due to overexpression of the cytoplasm-localized AOS, while (Z)-3-hexenal and (Z)-3-hexenyl acetate appeared not to be significantly altered. The data indicate that cytoplasmic AOS responds to wounding by increasing the levels of the wound-induced JA which in turn directly or indirectly enhances the expression of plant defense genes.