Marijana Krsnik-Rasol

Isoenzymes of peroxidase and esterase related to morphogenesis in Mammillaria gracillis Pfeiff. tissue culture

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 grown 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.

Quercetin influences response in Nicotiana megalosiphon infected by satellite-associated cucumber mosaic virus

Quercetin is a prominent flavonoid with a remarkable spectrum of biochemical activities. Its impact on human health, as well as its role in the normal development of plants has been exten-sively studied. In this work the influence of quercetin on satel-lite associated cucumber mosaic virus (CMV) infection and the response of Nicotiana megalosiphon host plants were investigated. Daily continuous flavonoid treatment of infected plants resulted in a significant increase in leaf mass and total protein synthesis. Attenuation of the yellowing symptom was also recorded, accompanied by lower expression of both viral and satellite replicative dsRna. This correlated with the decreased total peroxidase activity established in those plants. Expression of some glycoproteins (38 and 22.5 kDa), absent in healthy plants, were suppressed when infected plants were treated with quercetin. At the same time, some other infection-specific glycoproteins (22, 24 and 29 kDa) were significantly amplified by quercetin. The inability of quercetin to bind Cmv nucleoprotein or isolated ssRna was demonstrated. Quercetin has not shown any effect on Hsp70 synthesis in Cmv-infected plants.ZusammenfassungQuercetin ist ein bedeutendes Flavonoid mit einem bemer-kenswerten Spektrum biochemischer Aktivitäten. Seine Bedeutung für die menschliche Gesundheit, wie auch seine Funktion in der normalen pflanzlichen Entwicklung wurden weitgehend untersucht. In dieser Arbeit wurde der Einfluss von Quercetin auf die Abwehrantwort von Nicotiana megalo-siphon-Wirtspflanzen gegenüber Satelliten-assoziierten Gur-kenmosaikvirus (CMV)-Infektionen untersucht. Tägliche kon-tinuierliche Flavonoidbehandlung von infizierten Pflanzen resultierte in einem signifikanten Anstieg der Blattmasse und der gesamten Proteinsynthese. Eine Abschwächung der Ver-gilbungssymptome wurde ebenfalls verzeichnet, begleitet von geringerer Expression von replikativer Virus- und Satelliten-RNA. Dies korrelierte mit der Abnahme der Gesamtperoxidase-aktivität, die für diese Pflanzen nachgewiesen wurde. Einige Glycoproteine (38 und 22,5 kDa), die in gesunden Pflanzen nicht vorkommen, wurden unterdrückt, wenn die Pflanzen mit Quercetin behandelt wurden. Zur gleichen Zeit wurden einige andere infektionsspezifische Glycoproteine (22, 24 und 29 kDa) signifikant durch Quercetin verstärkt. Die Unfähigkeit von Quercetin, das CMV-Nukleokapsid oder isolierte ssRNA zu binden, wurde nachgewiesen. Quercetin zeigte keinen Effekt auf die HSP70-Synthese bei CMV-infizierten Pflanzen.

Spine micromorphology of normal and hyperhydric Mammillaria gracilis Pfeiff. (Cactaceae) shoots.

Artificial conditions of tissue culture affect growth and physiology of crassulacean acid metabolism plants which often results in formation of hyperhydric shoots. In in vitro conditions Mammillaria gracilis Pfeiff. (Cactaceae) growth switches from organized to unorganized way, producing a habituated organogenic callus which simultaneously regenerates morphologically normal as well as altered hyperhydric shoots. In this study, influence of tissue culture conditions on morphology of cactus spines of normal and hyperhydric shoots was investigated. Spines of pot‐grown Mammillaria plants and of in vitro regenerated shoots were examined with stereo microscope and scanning electron microscope. The pot‐grown plants had 16–17 spines per areole. In vitro grown normal shoots, even though they kept typical shoot morphology, had lower number of spines (11–12) and altered spine morphology. This difference was even more pronounced in spine number (six to seven) and morphology of the hyperhydric shoots. Scanning electron microscopy analysis revealed remarkable differences in micromorphology of spine surface between pot‐grown and in vitro grown shoots. Spines of in vitro grown normal shoots showed numerous long trichomes, which were more elongated on spines of the hyperhydric shoots; the corresponding structures on spine surface of pot‐grown plants were noticed only as small protrusions. Scanning electron microscopy morphometric studies showed that the spines of pot‐grown plants were significantly longer compared to the spines of shoots grown in tissue culture. Moreover, transverse section shape varies from elliptical in pot‐grown plants to circular in normal and hyperhydric shoots grown in vitro. Cluster and correspondence analyses performed on the scanning electron microscope obtained results suggest great variability among spines of pot‐grown plants. Spines of in vitro grown normal and hyperhydric shoots showed low level of morphological variation among themselves despite the significant difference in shoot morphology.

Protein glycosylation in sugar beet cell line can be influenced by DNA hyper- and hypomethylating agents

Protein glycosylation in sugar beet cell line can be influenced by DNA hyper- and hypomethylating agents Protein 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 vulgaris L.) 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.

Morphological and proteomic analyses of sugar beet cultures and identifying putative markers for cell differentiation

Normal (N), habituated nonorganogenic (HNO), and tumour (T) sugar beet cell lines were analysed in order to establish specific patterns of extracellular proteins and identify protein markers that might explain the distinct phenotypical 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 elongated, with reduced thylakoids and abundant phytoferritin deposits, while HNO plastids were small and vacuolated, with an irregular, underdeveloped thylakoid system. The extracellular 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.

In vitro conditions affect photosynthetic performance and crassulacean acid metabolism in Mammillaria gracilis Pfeiff. tissues

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 M. 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 β subunit of ATP synthase (β 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.

Estimation of Free and Bound MDA in Plant Extracts: Comparison Between Spectrophotometric and HPLC Methods

Lipid peroxidation is a complex process in which polyunsaturated fatty acids (PUFAs) are subjected to attack by oxygen-derived free radicals resulting in the formation of lipid hydroperoxides. In biological tissues, these lipid hydroperoxides are broken down into a variety of products including aldehydes and ketones [1]. The extend of lipid peroxidation is considered to be an important parameter for the identification of the oxidative stress [2]. The improvement of a technique for the measurement of this lipid peroxidation in living tissues is, consequently, of importance. A large variety of methods are available for the detection of lipid peroxidation products. These include determination of diene conjugation, lipid hydroperoxides, chemiluminescence, hydroxy acids, ethane and Thiobarbituric acid (TBA)-reactive materials. The estimation of TBA reactive products is the most widely used. The success of the TBA reaction method depends on the accuracy of the determination of MDA content. MDA is a three carbon dialdehyde which is widely produced in organisms as a end product of polyunsaturated lipid peroxidation. This method is of particular interest because of its simplicity and relatively good sensitivity, but it lacks specificity and often leads to overestimation due to other interfering compounds ([3],[4]). Treatment of biological sample with TBA under appropriate conditions results in the formation of pinkcolored products (λmax = 532 nm) and many substances which are not related to lipid peroxidation (sugars, amino acids, biliverdin, chemicals, glyoxal,...) may also form a TBA complex with significant absorbance around 530–535 nm.

Professor Zvonimir Devidé, on the occasion of his 90th birthday on August 6th 2011

Professor Zvonimir Devidé, on the occasion of his 90th birthday on August 6th 2011

Comparison of Fatty Acid Composition of Phospholipids in Normal and Habituated Sugar Beet Cell Lines

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.