Search

Effects of iso-osmotic concentrations of NaCl and mannitol were studied in Mammilaria gracilis (Cactaceae) in both calli and tumors grown in vitro. In both tissues, relative growth rates were reduced under osmotic stress, which were accompanied by a decrease in both tissue water and K+ content. However, growth was inhibited to a lesser extent after exposure to NaCl, when accumulation of Na+ ions was observed. In calli, only salinity increased proline content, whereas with tumors proline accumulated after both osmotic stresses. Osmotic stresses also induced oxidative damage in both cactus tissues, although higher oxidative injury was caused by mannitol in calli and by salt in tumors. Low iso-osmotic concentrations of NaCl (75 mM) and mannitol (150 mM) increased peroxidase, ascorbate peroxidase, and esterase activities, whereas elevated catalase activity was recorded only after mannitol treatment in both tissues. High osmotic stress generally decreased enzymatic activities. However, in calli, esterase activity increased in response to high salinity, whereas ascorbate peroxidase activity was enhanced after high mannitol stress. In conclusion, both in vitro-grown cactus tissues were found to be sensitive to osmotic stress caused by either mannitol or NaCl, but accumulation of Na+ ions in response to salt somewhat contributed to osmotic adjustment. However, more prominent oxidative damage induced by NaCl compared to mannitol in tumor could be related to ion toxicity. The mechanisms that mediate responses to salt- and mannitol-induced osmotic stresses differed and were dependent on tissue type.

Mammillaria gracilis Pfeiff. plants cultivated in the pot (pot plants, PP), as well as in vitro- grown normal shoots (NS), habituated callus (HC), hyperhydric shoots (HS), and tumour tissue (TT) were investigated in order to reveal the influence of in vitro culture on functionality of the photosynthetic apparatus and CAM photosynthesis in cactus Mammillaria gracilis Pfeiff. Photosynthetic pigments content as well as maximum (Fv/Fm) and effective (ΦPSII) quantum yield of photosystem II (PSII) decreased in all in vitro-grown tissues in comparison to PP. The decrease observed in hyperhydric HC, HS and TT correlated with a low expression of Rubisco large subunit (RbcL) and  subunit of ATP synthase ( ATP synt) and almost undetectable levels of protein D1, light- harvesting chlorophyll a/b-binding protein (LHCII) and cytochrome f protein of thylakoid Cyt b6/f- complex (Cyt f) found in these tissues. As for crassulacean acid metabolism (CAM) pattern, PP and NS expressed diurnal acid fluctuation, while HC, HS and TT failed to show it. Nevertheless, all M. gracilis tissues exhibited diurnal changes of phosphoenolpyruvate carboxylase (PEPC) activity indicating the typical CAM physiology. In conclusion, the photosynthesis was down regulated in all in vitro-grown tissues. NS maintained typical CAM photosynthesis, while HC, HS and TT withheld PEPC activity, but not acid accumulation specific for CAM. Minor changes observed in NS in comparison to PP could be attributed to the sugar supplementation while the more prominent deviations found in HC, HS and TT could be correlated with hyperhydricity and the loss of characteristic tissue organisation pattern.

Normal (N), habituated nonorganogenic (HNO), and tumour (T) sugar beet cell lines were analysed in order to establish specific patterns of extracellular proteins and iden- tify protein markers that might explain the distinct pheno- typical characteristics. Electron microscopy showed that the ultrastructure of N cells corresponds to that of parenchyma cells, and that these cells contain plastids with large starch grains. HNO and T cells had enlarged, lobed nuclei with an increased number of nucleoli ; the number of nuclei in HNO cells was greater than in T cells. The T plastids were elon- gated, with reduced thylakoids and abundant phytoferritin deposits, while HNO plastids were small and vacuolated, with an irregular, underdeveloped thylakoid system. The extra- cellular proteome of the cells was separated by sodium dodecyl sulphate polyacrylamide gel electrophoresis. Greater differences in protein expression were observed between the HNO and N lines than between the T and N lines. Sixteen of the most prominent bands differentially expressed among the cell lines were cut out from the gel and analyzed by mass spectrometry. Cell wall-modifying enzymes were identified, including a peroxidase whose expression was twofold higher in N and T tissue than in HNO tissue ; pectinesterase, which was expressed at a level threefold lower in the T line than in the other cell lines ; and xyloglucan endotransglucosylase, which was expressed at a level sixfold higher in HNO and T tissue. Three proteins belonged to the chitinase gene family and their expression was higher in HNO and T tissue than in N tissue. The differential expression of these proteins suggests that these play a role in cell line-specific cell wall composition and cell-to-cell adhesion.

Long term in vitro cultures of plant tissue spontaneously change at morphological, cellular and biochemical level. Horseradish tissues were cultivated in vitro for more than sixteen years. Primary crown-gall tumours were induced with Agrobacterium tumefaciens on leaf explants excised from plantlets propagated in vitro. Subcultured on hormone-free MS medium, transformed tissue continued to grow in an unorganised way (TN tumour) or as teratogenous tumour (TM teratoma) forming malformed shoots which never rooted. The aim of this study was to establish the morphological and biochemical status of this long term cultures. The study was performed by verifying biochemical marker of transformation and morphogenesis, which had been investigated when the culture was established (peroxidase, PPX) as well as by introducing new markers which have not been analysed so far in horseradish tissues (ascorbate peroxidase, APX ; phenylalanine-ammonia lyase, PAL ; and phenolics content). Morphological changes were observed only in teratoma tissue. Peroxidase activity and isoenzyme pattern indicated stability of horseradish tissue culture. The new biochemical markers were in a complete (PAL) and partial (APX) correlation with the PPX results and are therefore useful addition in biochemical characterisation of these tissues and can also be applied as markers of morphogenesis and tumorisation.

In this study cellular proteins of in vitro grown horseradish (Armoracia lapathifolia Gilib.) tissues of different morphologies, leaves (L), teratoma (TM) and tumor (TN), were separated by one-dimensional (1-DE) and two-dimensional polyacrylamide gel electrophoresis (2-DE) in order to reveal developmentally specific proteins. Moreover, proteins were subjected to analyses by lectin assays to reveal whether the morphological differences were reflected in protein glycosylation. 1-DE glycoprotein pattern showed substantial distinctions between horseradish tissues of different morphologies, which was in contrast to silver stained protein profiles, where only slightly qualitative changes were observed. 2-DE revealed more differences in expression of cellular proteins between examined tissues in comparison to 1-DE, while 2-DE blots confirmed quantitative difference in expression of 43 kDa glycoprotein between investigated tissues. Mass spectrometry analysis identified this protein as a typical horseradish peroxidase. Obtained results imply that the oligosaccharide structures could be important determinants of specificities of horseradish tissue morphology. (doi: 10.5562/cca2078)

Protein glycosylation in sugar beet cell line can be influenced by DNA hyper- and hypomethylating agentsProtein glycosylation is a co- and post-translational modification that influences protein function, stability and localization. Changes in glycoprotein pattern during differentiation/dedifferentiation events exist in animal cells and DNA methylation status is closely related to the changes. However, in plant cells this relationship is not yet established. In order to verify whether such a relation exists, hypermethylating drugs 2,4-dichlorophenoxyacetic acid and hydroxyurea, or hypomethylating drug 5-azacytozine were applied to sugar beet (Beta vulgarisL.) cells during 14 days of in vitro subculture, and the glycoprotein patterns of the cells were compared. The applied drugs were not toxic, as observed from cell phenotype and by measuring growth of the control and treated cells. Hyper and hypomethylating treatments influenced the activity of enzymes related to differentiation state of the cells: peroxidases and esterases, and their isoform patterns. Electrophoretic patterns of soluble and membrane proteins were similar between control and treatments, but the treatments modified N- and O-linked glycoprotein patterns as visible from GNA and PNA lectin blots. This suggested that hypermethylation and hypomethylation of genomic DNA in sugar beet cells affect protein glycosylation patterns and cellular metabolism, possibly in a mechanism similar to that existing in animal cells.

In vitro propagated plants of Mammillaria gracilis Pfeiff. (Cactaceae) develop calli without any exogenous growth regulators. This habituated tissue spontaneously regenerates morphologically normal as well as hyperhydric shoots. In this study, a possible involvement of activated oxygen metabolism in habituation and hyperhydricity in in vitro propagated plants of Mammillaria gracilis Pfeiff. (Cactaceae) was investigated. Significantly higher malondialdehyde (MDA) and carbonyl contents as well as hydrogen peroxide (H2O2) production were observed in habituated callus (HC), hyperhydric regenerated shoots (HS), and tumors (TT) in comparison to normal regenerated shoots (NS). Lipoxygenase (LOX) activity showed a similar trend, with a clear increase in activity in HC and HS. The activities of antioxidative enzymes, namely, peroxidase (POX), ascorbate peroxidase (APX), and catalase (CAT), were also higher in HC, HS, and TT, whereas an increase in superoxide dismutase (SOD) activity was observed in HC and HS. The majority of antioxidative isoenzymes were common to all cactus tissues, although a few tissue-specific bands were noticed. Significant decreases in phenylalanine ammonia lyase (PAL) activity, total phenolic content, and lignification were found in HS, HC, and TT in comparison to NS. Our results showed the appearance of a prominent oxidative stress in HC, HS, and TT as well as a strong induction of the antioxidant system indicating that activated oxygen metabolism could be involved in habituation and hyperhydricity and linked to the loss of tissue organization in M. gracilis.

Summary In vitro propagated plants of the cactus Mammillaria gracillis Pfeiff. ( Cactaceae ) spontaneously produced callus. The habituated callus regenerated normal and hyperhydric shoots without the addition of growth regulators. Tumours were obtained by infecting cactus explants with Agrobacterium tumefaciens ; the wild strain B6S3 (tumour TW) or with the rooty mutant GV3101 (tumour TR). Both tumour lines grew vigorously, never expressing any morphogenic potential. In this study, cactus shoots, callus, normal and hyperhydric regenerants and TW and TR tumours were compared with regard to peroxidase (EC 1.11.1.7) and esterase activity, and isoenzyme patterns. Guaiacol peroxidase activity was the lowest in the cactus shoots and in the normal regenerants. Callus, hyperhydric regenerants and tumours had peroxidase activity of 6 to 7 times higher. Esterase activity was measured with 1- and 2-naphthylacetate as broad-spectrum substrates. The highest esterase activity was determined in tumours with both substrates. All tissues, except the TR tumour, had higher esterase activity for 2- compared to 1-naphtylacetate. Peroxidase and esterase isoenzyme patterns were not completely identical among the investigated tissues.

Normal, habituated and transformed in vitro tissue lines of sugar beet (Beta vulgaris L.), horseradish (Armoracia lapathifolia Gilib.) and potato (Solanum tuberosum L.) were studied with regard to isoesterase patterns. Isoenzymes were separated in gradient gels (5-12%) of polyacrylamide and by isoelectric focussing in pH range 4-9. 1- and 2-naphtylacetate were used as substrates of broad spectrum which cover also esterases (arylesterases and carboxylesterases) reacting with organophosphorous compounds. Distinct isoesterase patterns were noticed in sugar beet normal, habituated and crown gall tumour tissues. Horseradish tumour and teratoma, on the contrary, differed only in one anodic isoenzyme. Even the malformed shoots and unorganised tissue of teratoma had the same patterns. In potato tuber tissue, change in isoesterase pattern, characterised by disappearance of a dominant dark area, was observed during tumour development. The gradient gels gave more stable and reproducible isoenzyme patterns than isoelectric focussing.

The composition of the lipid bilayer of biological membranes is of importance for the properties of the membrane and consequently for the function of the cell. The biomembranes are dynamic entities. Both the protein and lipid components of the membrane are constantly being turned over [1], [2] so as to maintain its functionnal state. Various features of the membrane lipids such as the composition, degree of unsaturation and surface charge properties affect and regulate the activities of membrane proteins. Hormones have also been implicated in the turnover of membrane lipids and protein components [3], [4]. Thus, the membrane properties are subject to very precise regulation for the maintenance of homeostasis. Habituated cells are considered to be the result of neoplasic progression of plant cells due to in vitro conditions and they exhibit many cytological and biochemical characteristics of animal tumors [5]. The role of membrane lipid alterations in the biological behavior of animal tumor cells have been investigated by several laboratories and some relations between altered membrane lipids and neoplasia have been underlined. Important membrane alterations have been reported in fatty acid saturation and phospholipid composition during hepatocarcinogenesis [6]. As membrane phospholipids are supposed to function as precursors of bioactive substances [7], enzyme activators [8] and modulators of plasma membrane-bound ATPases [9], we have investigated phospholipid content and their fatty acid composition in normal (auxin and cytokinin requiring) and habituated (auxin and cytokinin independent) sugarbeet calli.