Enhanced salt stress tolerance in transgenic potato plants (<Emphasis Type="Italic">Solanum tuberosum</Emphasis> L.) expressing a bacterial <Emphasis Type="Italic">mtl</Emphasis>D gene

Bacterial mannitol 1-phosphate dehydrogenase (mtlD) gene was introduced into potato (Solanum tuberosum L.) by Agrobacterium tumefaciens-mediated transformation. Transgenic plants were selected on a medium containing 100 mg l⁻¹ kanamycin and confirmed by polymerase chain reaction (PCR), Southern blotting, and RT-PCR analyses. All of the selected transformants accumulated mannitol, a sugar alcohol that is not found in wildtype potato. Experiments designed for testing salt tolerance revealed that there was enhanced NaCl tolerance of the transgenic lines both in vitro and in hydroponic culture. Compared to 0 mM NaCl, the shoot fresh weight of wildtype plants was reduced by 76.5% at 100 mM NaCl under hydroponic conditions. However, under the same condition, the shoot fresh weight of transgenic plants was reduced only by 17.3%, compared to 0 mM NaCl treatment. The improved tolerance of this transgenic line may be attributed to the induction and progressive accumulation of mannitol in the roots and shoots of the plants. In contrast to in vitro experiments, the mannitol content in the transgenic roots and shoots increased at 50 mM NaCl and decreased slightly at 75 and 100 mM NaCl, respectively. Overall, the amount of accumulated mannitol in the transgenic lines was too small to act as an osmolyte; thus, it might act as an osmoprotectant. However, the results demonstrated that mannitol had more contribution to osmotic adjustment in the roots (but not in shoots). Finally, we concluded that mtlD expression in transgenic potato plants can significantly increase the mannitol accumulation that contributes to the enhanced tolerance to NaCl stress. Furthermore, although this enhanced tolerance resulted mainly from an osmoprotectant action, an osmoregulatory effect could not be ruled out.     

Detection of peroxisomal proteins and their mRNAs in serial sections of fetal and newborn mouse organs.

We present a protocol for detection of peroxisomal proteins and their corresponding mRNAs on consecutive serial sections of fetal and newborn mouse tissues by immunohistochemistry (IHC) and nonradioactive in situ hybridization (ISH). The use of perfusion-fixation with depolymerized paraformaldehyde combined with paraffin embedding and digoxigenin-labeled cRNA probes provided a highly sensitive ISH protocol, which also permitted immunodetection with high optical resolution by light and/or fluorescence microscopy. Signal enhancement was achieved by the addition of polyvinyl alcohol (PVA) for ISH color development. For IHC, signal amplification was obtained by antigen retrieval combined with biotin-avidin-HRP and Nova Red as substrate or by the catalyzed reporter deposition of fluorescent tyramide. Using this protocol, we studied the developmental changes in localization of the peroxisomal marker enzymes catalase (CAT) and acyl-CoA oxidase 1 (AOX), the key regulatory enzyme of peroxisomal beta-oxidation, at the protein and mRNA levels in mice from embryonic Day 14.5 to birth (P0.5). The mRNA signals for CAT and AOX were detected in sections of complete fetuses, revealing organ- and cell-specific variations. Here we focus on the localization patterns in liver, intestine, and skin, which showed increasing mRNA amounts during development, with the strongest signals in newborns (P0.5). Immunolocalization of the corresponding proteins revealed, in close correlation with the mRNAs, a distinct punctate staining pattern corresponding to the distribution of peroxisomes. (J Histochem Cytochem 49:155-164, 2001)     

The role of 15-lipoxygenase in disruption of the peroxisomal membrane and in programmed degradation of peroxisomes in normal rat liver.

Our earlier electron microscopic observations revealed that prolonged exposure of glutaraldehyde-fixed rat liver sections to buffer solutions induced focal membrane disruptions of peroxisomes with catalase diffusion as shown cytochemically. Recently, it was suggested that 15-lipoxygenase (15-LOX) might be involved in natural degradation of membrane-bound organelles in reticulocytes by integrating into and permeabilizing the organelle membranes, leading to the release of matrix proteins. We have now investigated the localization of 15-LOX and its role in degradation of peroxisomal membranes in rat liver. Aldehyde-fixed liver slices were incubated in a medium that conserved the 15-LOX activity, consisting of 50 mM HEPES-KOH buffer (pH 7.4), 5 mM mercaptoethanol, 1 mM MgCl(2), 15 mM NaN(3), and 0.2 M sucrose, in presence or absence of 0.5-0.05 mM propyl gallate or esculetin, two inhibitors of 15-LOX. The exposure of aldehyde-fixed liver sections to this medium induced focal disruptions of peroxisome membranes and catalase diffusion around some but not all peroxisomes. This was significantly reduced by both 15-LOX inhibitors, propyl gallate and esculetin, with the latter being more effective. Double immunofluorescent staining for 15-LOX and catalase revealed that 15-LOX was co-localized with catalase in some but not all peroxisomes in rat hepatocytes. By postembedding immunoelectron microscopy, gold labeling was localized on membranes of some peroxisomes. These observations suggest that 15-LOX is involved in degradation of peroxisomal membranes and might have a physiological role in programmed degradation and turnover of peroxisomes in hepatocytes. (J Histochem Cytochem 49:613-621, 2001)     

Induction of tubular peroxisomes by UV irradiation and reactive oxygen species in HepG2 cells.

Peroxisomes in the human hepatoblastoma cell line HepG2 exhibit a high degree of plasticity. Whereas in confluent cultures they appear as small (0.1-0.3 micrometer) spherical particles, they undergo dramatic changes, forming elongated tubules measuring up to 5 micrometer on separation of cells and cultivation at low density. We recently showed that several growth factors, including nerve growth factor (NGF), induce the formation of tubular peroxisomes and that this induction is sensitive to K 252b, a specific tyrosine kinase inhibitor, suggesting the involvement of this signal transduction pathway. Because tyrosine kinase is also involved in signal transduction via the reactive oxygen species (ROS), we have analyzed in this study the effects of UV irradiation, H(2)O(2), and oxygen on tubulation of peroxisomes. UVC irradiation induced a significant increase in formation of tubular peroxisomes (40-50% of cells) and this effect was dose-dependently inhibited by pretreatment with N-acetyl cysteine, confirming the involvement of ROS in the UV effect. Furthermore, H(2)O(2) also directly induced the tubulation of peroxisomes, although to a lesser extent. Finally, cultivation under hypoxic conditions (1.5% O(2)) drastically reduced the inducing effect of fetal calf serum on tubulation of peroxisomes, suggesting the involvement of oxygen-mediated signaling. Taken together, our observations indicate that ROS induce the tubulation of peroxisomes in HepG2 cells. Because peroxisomes harbor most of the enzymes for catabolism of ROS, the tubulation and expansion of the peroxisome compartment could have a cell rescue function against the destructive effects of ROS.     

The importance of tissue fixation for light microscopic immunohistochemical localization of peroxisomal proteins: the superiority of Carnoy's fixative over Baker's formalin and Bouin's solution

We have compared the effects of fixation with three commonly used fixatives upon preservation of the antigenicity of six peroxisomal proteins in rat liver using both immunohistochemical staining and Western blotting of fixed tissue extracts. The immunoreactivity of all six peroxisomal proteins was well preserved and peroxisomes were clearly identified in material fixed in Carnoy's fixative. Moreover, the corresponding proteins stained well in Western blots prepared from extracts of Carnoyfixed material. The intensity of the immunohistochemical staining was reduced at different rates for individual peroxisomal proteins after fixation in Baker's formalin, but peroxisomes were still well visualized with antibodies to catalase and some -oxidation enzymes. No evidence of immunohistochemical staining for any peroxisomal antigens was obtained after fixation in Bouin's fluid. For detection of the antibody binding sites in Carnoy's fixed material, the avidin-biotin-peroxidase complex (ABC) with aminoethyl carbazole as chromogen was found to be superior to the methods of peroxidase-antiperoxidase/diaminobenzidine and protein A-gold with silver intensification. Using Carnoy-fixative and the ABC-method, we demonstrate light microscopic immunohistochemical localization of peroxisomal antigens in several rat tissues as well as in human post-mortem liver.     

Evidence that glucagon stabilizes rather than activates mitochondrial functions in rat liver

The present study is concerned with the question as to whether the acute treatment of intact rats or hepatocytes with glucagon and dibutyryl cAMP, respectively, leads to a stabilization or an activation of mitochondrial functions, such as state-3 respiration, succinate dehydrogenase activity and pyruvate carboxylase activity. For this purpose, the influence of various parameters of mitochondria preparation (isolation medium, washing steps, storage) as well as of phospholipase A inhibitors (cinchocain, chloroquine) on the expression of the hormone effect was examined. With regard to the above mentioned functions, the values displayed by control mitochondria were found to be considerably higher if mannitol instead of sucrose had been used for isolation. Accordingly, only small effects of hormone treatment became apparent. The addition of cinchocain or chloroquine to the sucrose medium yielded results similar to those obtained with mannitol. Furthermore, the hormone effect on state-3 respiration and succinate dehydrogenase activity was only small if the mitochondria had been prepared faster than usual and had been used without washing. Regarding pyruvate carboxylase, a considerably smaller glucagon effect was observed when it was assayed at 25 degrees C and not (as usual) at 37 degrees C. Our results indicate that glucagon application stabilizes rather than activates mitochondrial functions.     

Selective cytochemical localization of peroxidase, cytochrome oxidase and catalase in rat liver with 3,3'-diaminobenzidine

In rat liver, three different enzymes with peroxidatic activity are demonstrated with modifications of the DAB-technique: peroxidase in the endoplasmic reticulum of Kupffer cells, catalase in peroxisomes and cytochrome oxidase in mitochondria. The major problem of the DAB-methods is their limited specificity so that often in tissues incubated for one enzyme the other two proteins are also stained simultaneously. We have studied the conditions for selective staining of each of these three enzymes in rat liver fixed either by perfusion with glutaraldehyde or by immersion in a modified Karnovsky's glutaraldehyde-formaldehyde fixative. The observations indicate that in perfusion fixed material selective staining can be obtained by reduction of the incubation time (5 min) and the use of optimal conditions for each enzyme. In livers fixed by immersion the distribution of the staining is patchy and irregular and usually longer incubation times (15-30 min) are required. Selective staining of peroxidase in Kupffer cells was obtained by brief incubation at room temperature in a medium containing 2.5 mM DAB in cacodylte buffer pH 6.5 and 0.02% H2O2. The exclusive staining for cytochrome oxidase in cristae of mitochondria was achieved after short incubation in 2.5 mM DAB in phosphate buffer pH 7.2 containing 0.05% cytochrome c. For selective demonstration of catalase in peroxisomes the tissue was incubated in 5 mM DAB in Teorell-Stenhagen (or glycine-NaOH) buffer at pH 10.5 and 0.15% H2O2. The prolongation of the incubation time in peroxidase medium caused marked staining of both mitochondria and peroxisomes. In the cytochrome oxidase medium longer incubations led to slight staining of peroxisomes. The catalase medium was quite selective for this enzyme so that even after incubation for 120 min only peroxisomes stained.     

Intracellular distinction between peroxidase and catalase in exocrine cells of rat lacrimal gland: A biochemical and cytochemical study

Summary The lacrimal gland (Glandula orbitalis externa) of rat contains both peroxidase and catalase and was used as a model for biochemical and cytochemical distinction between peroxidase and catalase. Both enzymes were isolated by ammonium sulfate precipitation from tissue homogenates, and the effects of fixation with glutaraldehyde and various conditions of incubation were investigated colorimetrically using DAB as hydrogen donor. The lacrimal gland peroxidase is strongly inhibited by glutaraldehyde treatment. In contrast, for catalase the fixation with glutaraldehyde is the prerequisite for demonstration of its peroxidatic activity. The maximal peroxidatic activity was obtained after treatment of catalase with 3% glutaraldehyde, higher concentrations being inhibitory. For lacrimal gland peroxidase, the maximal rate of oxidation of DAB is at pH 6.5, whereas for catalase it is at pH 10.5. The optimal concentration of H₂O₂ for lacrimal gland peroxidase is at 10⁻³ M and for peroxidatic activity of catalase at 10⁻¹ M. These optimal conditions obtained biochemically were applied to tissue sections of rat lacrimal gland. After the fixation of tissue with a low concentration of glutaraldehyde and incubation in the DAB medium at neutral pH containing 10⁻³ M H₂O₂ (Peroxidase medium), the reaction product was localized in the cisternae of the rough endoplasmic reticulum, in elements of the Golgi apparatus, and in secretory granules. After the fixation of tissue with 3% glutaraldehyde and incubation in the DAB-medium containing 10⁻¹ M H₂O₂ and at pH 10.5 (catalase medium), the staining in the endoplasmic reticulum, the Golgi-apparatus and in secretory granules was completely inhibited and reaction product was localized exclusively in small (0.2–0.5 μ) particles similar to small peroxisomes described in various other cell-types. This work was presented in part at the twenty-fifth Annual Meeting of the Histochemical Society, April 5–6, 1974. Atlantic City, N.J., J. Histochem. Cytochem.22, 288 (1974).     

Isolation and characterization of peroxisomes from the liver of normal untreated rats

The classic method of Leighton et al. [(1968) J. Cell Biol. 37, 482-513] for the isolation of peroxisomes from rat liver involves the use of Triton WR-1339 which alters the biochemical properties of this organelle and requires the specialized type Beaufay-rotor which is not easily available. We have employed Metrizamide as the gradient medium and a commercial type vertical rotor to obtain highly purified and structurally well-preserved peroxisomes from normal untreated animals. The livers were homogenized in buffered 0.25 M sucrose and a slightly modified 'light mitochondrial fraction' was prepared by differential centrifugation. This was loaded on top of a linear Metrizamide gradient (1.12-1.26 g/cm3) and subjected to an integrated force of 1.252 X 10(6) X (g X min) using a Beckman VTi 50 vertical rotor. Peroxisomes banded at the density of 1.245 g/cm3. In the isolated fraction 95% of the protein was contributed by peroxisomes, which exhibited a strong activity for cyanide-insensitive lipid beta-oxidation. The purity of fractions was also confirmed by morphometry, which revealed that 98% of isolated particles consisted of peroxisomes. The latency for catalase was about 90% indicating a high degree of peroxisomal integrity. This corresponded to the low level of extraction of catalase in 3,3'-diaminobenzidine-stained filter preparations. The entire procedure took about five hours. Highly purified and structurally well preserved peroxisomes should be useful in further elucidation of the function of this organelle and especially in studies of peroxisomal enzymes with multiple intracellular localizations.