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Salt and drought stress are important abiotic factors that negatively affect plant growth and yield. To understand how these stress factors affect metabolism at the cellular level, we analyzed cation concentrations and expression of cellular and extracellular proteins, as well as their functions and types. Cells of the industrially important halophyte sugar beet were exposed to 300 mM NaCl and 600 mM mannitol as stressors in modified Gamborg B5 liquid medium (PG0). Severe stress altered the intracellular concentrations of the most measured cations. The cellular proteome revealed that both stressors provoked significant differential regulation of 110 cellular proteins. About 80% of the identified proteins were classified in metabolism, energy, or cell rescue, defense and virulence categories. We identified several novel proteins that respond to stress, including a member of the bZIP family of transcription factors, a member of the glycine-rich RNA-binding proteins, and the K+ channel beta subunit. Among extracellular proteins we found previously unreported stress-responsive proteins, a beta-xylosidase and an isoform of chitinase. The obtained results indicate that salt and drought stress disturbed the concentrations of cellular cations and the affected expression of cellular and extracellular proteins in sugar beet cells.

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.

Summary 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.

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)

Plant tissues contain relatively low amounts of proteins whose extraction is often difficult due to the presence of interfering compounds such as rigid cellulosic cell wall, storage polysaccharides, lipids and other contaminants that can cause protein degradation or modification. Therefore it is important to optimize protein extraction and to establish a robust protocol for two-dimensional gel electrophoresis (2-DE) and downstream processing. In this study, acetone, trichloroacetic acid/acetone and Tris-buffered phenol/methanol extraction protocols were evaluated on non-model and recalcitrant plants: a Beta vulgaris L. in vitro cell line, Mammillaria gracilis Pfeiff. in vitro plants and Sempervivum tectorum L. leaves. Although phenol extraction was more time-consuming than the other two methods, it gave almost two-fold higher protein yield, and spectral analysis showed less contamination. SDS-PAGE, 2-DE and bioinformatic analysis showed that protein extraction using phenol was superior to the other two methods, providing more protein bands or spots on the gels and less proteolysis. These results lead to the conclusion that the phenol method is the most suitable protein extraction method for these non-model and recalcitrant plant tissues. (doi: 10.5562/cca1804)

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.

Plant development is influenced by changes in the levels and types of sugars produced metabolically. The normal (N), habituated organogenic (HO) and habituated nonorganogenic (HNO) sugar beet cell lines originate from the same mother plant but exhibit distinct levels of morphogenesis and differentiation, and contain different levels of simple carbohydrates. We aim to elucidate whether differences in the abundance and activity of enzymes involved in carbohydrate metabolism and sugar sensing/signalling help explain the different carbohydrate profiles and differentiation states of the cell lines. Using 13C NMR spectroscopy to analyze cultures of the cell lines over 28 days, we found that N cells accumulated sucrose; HO cells sucrose, glucose and fructose; and HNO cells glucose and fructose. Of three invertase isoforms, the activity of cell wall invertase (CWI) was highest in all the cell lines, and CWI activity was greatest in HNO line. The specific accumulation of intracellular carbohydrates during subculture correlated strongly with CWI activity but less so with the vacuolar and cytoplasmic invertase isoforms, or with sucrose synthase activity. Cell lines showed differences in how sugars regulated invertase and sucrose synthase activity. The role of sugar sensing in the regulation of CWI activity was investigated in the cell lines using glucose and sucrose, as well as carbohydrate analogues such as mannitol, 2-O-deoxyglucose and 3-O-methylglucose. Differences in the regulation of CWI activity by carbohydrates across the three cell lines suggest that CWI can be fine-tuned according to the specific carbohydrate requirements of each line during growth. Differences in sugar signalling pathways across the cell lines were explored using glucose and sucrose in the presence of inhibitors of protein kinases or phosphatases. Taken together, our findings suggest that specific regulation of CWI activity plays an important role in determining the intracellular carbohydrate levels of sugar beet cell lines, and possibly their differentiation state as well., Na razvoj biljke utječu promjene razine i tipa proizvedenog šećera. Iako sve stanične linije šećerne repe, i to normalna, prilagođena organogena i prilagođena neorganogena potječu od iste biljke, na različitim su stupnjevima diferencijacije i morfogeneze te sadrže različitu količinu jednostavnih ugljikohidrata. Svrha je rada bila utvrditi može li se razlikama u količini i aktivnosti enzima, koji sudjeluju u metabolizmu šećera te onih u detekciji i prijenosu signala šećera, objasniti razlika u stupnju diferencijacije staničnih linija te u udjelu ugljikohidrata u njima. Primjenom 13C NMR spektroskopije analizirane su stanične linije tijekom 28 dana kultivacije i ustanovljeno je da normalne stanične linije akumuliraju saharozu, prilagođena organogena stanična linija saharozu, glukozu i fruktozu, a prilagođena neorganogena stanična linija šećerne repe glukozu i fruktozu. Od 3 izoformna oblika invertaze najveća je aktivnost invertaze stanične stijenke u sve 3 stanične linije, osobito u prilagođenih neorganogenih stanica. Specifičnost akumulacije ugljikohidrata u stanici tijekom kultivacije uvelike je ovisila o aktivnosti invertaze stanične stijenke, a manje o onoj vakuolarne i citoplazmatske invertaze te saharoza sintetaze. Šećeri su različito regulirali aktivnost invertaze stanične stijenke i saharoza sintetaze, ovisno o tipu stanične linije. Njihova je uloga istražena pomoću glukoze i saharoze te njihovih analoga, kao što su manitol, 2-O-deoksiglukoza i 3-O-metilglukoza. Utvrđeno je da se aktivnost invertaze stanične stijenke može fino regulirati pomoću ugljikohidrata, ovisno o potrebi staničnih linija za izvorom šećera tijekom rasta. Razlike u putovima prijenosa signala šećera u staničnih linija istražene su pomoću glukoze i saharoze u prisutnosti inhibitora protein kinaza i fosfataza. Na kraju, rezultati autora potvrđuju da specifična regulacija aktivnosti invertaze stanične stijenke ima važnu ulogu u određivanju razine ugljikohidrata u staničnim linijama šećerne repe, a možda i stupnju diferencijacije stanica.

In a previous study it was reported that transformed tissue of horseradish (Armoracia lapathifolia Gilib.), obtained by infection of leaf explants with A. tumefaciens, developed two tumour lines with different morphology. One line grew as a completely unorganized tissue (TN – tumour tissue), while the other line grew as a partially organized teratogenous tumour with malformed hyperhydric shoots (TM – teratoma tissue), but did not regenerate the whole plant of normal morphology. The factor responsible for this problem could be the increased production of reactive oxygen species (ROS). Therefore, in this study a possible involvement of activated oxygen metabolism in dedifferentiation and hyperhydricity in TM and TN tissues is investigated. Elevated values of malondialdehyde and protein carbonyl contents found in TM and TN, in comparison with plantlet leaf, confirm the presence of oxidative stress. However, lower H2O2 content was measured in TM and TN. Lipoxygenase (LOX) activity was more pronounced in TM and especially in TN compared to leaf, which suggests that the LOX-dependent peroxidation of fatty acids might be one of the causes of oxidative damage. Moreover, significantly higher peroxidase (PRX) and ascorbate peroxidase (APX) activity as well as the increased number of their isoforms was found in transformed TM and TN in comparison with leaf. On the other hand, significantly lower superoxide dismutase (SOD) activity was found in TM and TN, which correlates with lower H2O2 content. High catalase (CAT) activity measured in leaf and partially organized TM is consistent with the role of CAT in growth and differentiation. In conclusion, in horseradish transformed tissues that underwent dedifferentiation and hyperhydricity, prominent oxidative damage was found. This result suggests that oxidative stress could be associated with the inability of partially organized teratogenous TM to regenerate plantlets with normal morphology., Dosadašnja istraživanja autora pokazala su da je transformirano tkivo hrena (Armoracia lapathifolia Gilib.), nakon infekcije bakterijom A. tumefaciens, razvilo dvije morfološki različite linije tumorskog tkiva. Jedna je linija rasla kao potpuno neorganizirano tumorsko tkivo, a druga kao djelomično organizirani teratogeni tumor s malformiranim hiperhidriranim izdancima (teratomska linija), ali bez mogućnosti regeneriranja biljke normalnih morfoloških karakteristika. Budući da bi povećana proizvodnja reaktivnih oblika kisika mogla uzrokovati promjenu morfoloških karakteristika, u ovom je radu istraživan mogući utjecaj kisika na dediferencijaciju i hiperhidriranost u potpuno neorganiziranom tumorskom tkivu i teratomskoj liniji. Uspoređujući povećane vrijednosti malondialdehida i proteinskih karbonila u potpuno neorganiziranom tumorskom tkivu i teratomskoj liniji s onima u listu hrena, uočena je pojava oksidacijskog stresa iako je izmjeren manji udjel H2O2. Aktivnost lipoksigenaze povećana je u teratomskoj liniji, a još više u potpuno neorganiziranom tumorskom tkivu, što pokazuje da bi peroksidacija masnih kiselina ovisna o aktivnosti lipoksigenaze mogla biti jedan od uzroka oksidacijskoga stresa. Štoviše, u transformiranim je tkivima za razliku od lista uočena bitno veća aktivnost peroksidaze i askorbat peroksidaze te veći broj njihovih izoenzima. S druge strane, izmjerena je kudikamo manja aktivnost superoksid dismutaze, s obzirom na smanjeni udjel H2O2. Veća aktivnost katalaze u listu i teratomskoj liniji povezana je s ulogom katalaze u rastu i diferencijaciji. Dobiveni rezultati potvrđuju prisutnost oksidacijskog stresa u dediferenciranim i hiperhidriranim tumorskim tkivima hrena, odnosno da je oksidacijski stres posljedica nesposobnosti teratomske linije da regenerira biljke normalne morfologije.

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

The demand for natural food colourants is increasing because of public awareness of their health benefits. Betalains are nitrogen-containing plant pigments whose colours range from red-violet betacyanins to yellow betaxanthins. They are used for colouring dairy products, meat and frozen desserts. Betalains have attracted additional interest because of their antioxidative, anti-inflammatory and anticarcinogenic properties. The main source of commercially produced betalains is red beet root, but alternative sources are found in plants from the Amaranthaceae and Cactaceae families. Another alternative source is plant cell culture in bioreactors, although optimization of pigment production seems necessary. In this paper we synthesize the results of recent studies on betalain biosynthesis, chemical properties, sources, biotechnology and applications., Sve je veća potražnja za prirodnim bojilima zbog njihovih zdravstvenih pogodnosti. Betalaini su biljni pigmenti koji sadrže dušik, s rasponom boja od crveno-ljubičastih betacijana do žutih betaksantina. Koriste se za bojanje mliječnih proizvoda, mesa i smrznutih deserta. Zanimljivi su i zbog svoga antioksidativog, protuupalnog i antikancerogenog djelovanja. Glavni komercijalni izvor betalaina je korijen cikle, a alternativni izvori pigmenta nalaze se u biljkama iz porodica Amaranthaceae i Cactaceae. Biljne stanice uzgajane u bioreaktorima također su izvor pigmenata iako je potrebno daljnje optimiranje njihove proizvodnje. U ovom su radu sažeti rezultati nedavnih istraživanja biosinteze betalaina, njihovih kemijskih svojstava, izvora, biotehnologije i uporabe.

The extracellular proteins in three distinctly induced embryogenic lines of pumpkin (Cucurbita pepo L.) cultivated in four MS media modified regarding the nitrogen composition or auxin presence/absence have been analyzed. Extracellular glycoproteins containing α-D-mannose were specifically detected by the lectine concavalin A. During the cultivation of embryogenic tissue in the medium supplemented with reduced nitrogen, the embryos were mostly arrested at preglobular and globular developmental stages, which coincide with the absence of protein secretion. Secreted glycoproteins of 76, 68, 37 and 34 kDa were detected only if any of the three lines were cultivated in the medium that stimulates embryo development, irrespectively of the addition of 2,4-dichlorophenoxyacetic acid or tunicamycin. The glycoprotein of 64 kDa was detected in all lines cultivated in hormone-free MS medium with conventional nitrogen sources and it appears to be associated with embryo maturation. Tunicamycin treatment did not influence embryogenesis, although it specifically affected glycosylation of proteins in the investigated lines. Our results show that besides auxin, the source of nitrate is of great importance for proper protein glycosylation, excretion and developmental transition of pumpkin somatic embryos., Analizirani su izvanstanični proteini tri ustaljene embriogene linije bundeva (Cucurbita pepo L.) koje su uzgajane na četiri različite podloge MS s obzirom na udjel dušika i prisutnost/odsutnost auksina. Izvanstanični proteini što sadrže α-D-manozu specifično su dokazani lektinom konkavalinom A. Tijekom uzgoja embriogenoga tkiva na podlozi s dodatkom reduciranog dušika, razvitak većine embrija bio je zakočen u predglobularnom i globularnom razvojnom stadiju, što se podudaralo s nedostatkom izlučivanja proteina. Izlučivanje proteina od 76, 68, 37 i 34 kDa dokazano je u sve tri linije tijekom uzgoja na podlozi koja pospješuje razvoj embrija, neovisno o dodatku 2,4-diklorofenoksioctene kiseline ili tunikamicina. Glikoprotein od 64 kDa utvrđen je u sve tri linije uzgajane na podlozi MS bez dodatka hormona i s nepromijenjenim izvorima dušika, te se mogao povezati sa sazrijevanjem embrija na ovoj podlozi. Izlaganje tunikamicinu nije utjecalo na embriogenezu, iako je imalo specifično djelovanje na glikozilaciju proteina ispitanih linija. Rezultati pokazuju da osim auksina i izvori dušika imaju važnu ulogu u pravilnoj glikozilaciji proteina, njihovom izlučivanju i dinamici razvitka somatskih embrija bundeve.

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.

The objective of this work is to transform sugar beet (Beta vulgaris L. var. altissima) cells using Agrobacterium tumefaciens, a wild octopine strain B6S3, and to study metabolic changes associated with the transformation. From the primary tumours on leaf fragments, two tumour lines were established: one pale green and the other red-violet in colour. The red-violet pigment was identified by means of high-performance liquid chromatography as betanin. Betanin production is strongly light-dependent. To enhance the betanin yield, nutrient media with different carbohydrates like sucrose, a combination of glucose and fructose, or only glucose or only fructose were tested. The selection of carbohydrate affected betanin production and yield. It was observed that the production of betanin per g of dry mass was 20–40 % higher on simple carbohydrates, especially fructose, than on sucrose or the combination of glucose and fructose. However, due to higher biomass production on the medium with sucrose, the highest total yield of betanin was obtained in the presence of sucrose. The most suitable time for possible betanin extraction was between days 7 and 14 for tissue grown on sucrose. The tumour line seems to be promising as an alternative source of betanin as well as a model to study sugar-regulated genes involved in tissue morphology control., Svrha je rada bila transformirati stanice šećerne repe (Beta vulgaris L. var. altissima) bakterijom divljeg soja B6S3 Agrobacterium tumefaciens, te proučavati metaboličke promjene nakon transformacije. Od primarnih tumora koji su se razvili na zaraženim listovima, uspostavljene su dvije tumorske linije: svijetlozelena i crveno-ljubičasta. Pigment iz crvene linije identificiran je visokotlačnom tekućinskom kromatografijom kao betanin. Proizvodnja betanina ovisila je o svjetlosti. Da bi se pojačao prinos betanina, ispitana je hranidbena podloga s različitim šećerima, i to: saharozom, glukozom i fruktozom te samo s glukozom ili samo s fruktozom. Proizvodnja i prinos betanina ovisili su o dodanom šećeru. Proizvodnja betanina po gramu suhe tvari bila je 20-40 % viša na monosaharidima, posebice fruktozi, nego na saharozi ili kombinaciji glukoze i fruktoze. Međutim, zbog obilnije biomase, najveći prinos betanina ostvaren je na podlozi sa saharozom. Najpovoljnije vrijeme za iskorištavanje betanina je između prvog i drugog tjedna supkulture na saharozi. Zaključno, dobivena tumorska linija prikladan je alternativni izvor pigmenta betanina, a i model za istraživanje gena koji uz pomoć šećera regulira morfologiju biljnih tkiva.

In vitro grown plantlets, tumour and teratoma tissues of horseradish (Armoracia lapathifolia Gilib.) were compared with regard to glucosinolate profiles. Plantlets produced significantly higher amounts of total glucosinolates than tumour and teratoma tissues. The aliphatic glucosinolate sinigrin was quantitatively dominant. Plantlets also contained lower amounts of an aromatic glucosinolate, gluconasturtiin and indole compounds: glucobrassicin, 4-methylglucobrassicin and 4-hydroxyglucobrassicin. In tumour and teratoma tissues only these indole glucosinolates were detected. The activity of enzyme myrosinase (β-thioglucosidase) was significantly higher in plantlets than in teratoma. No myrosinase activity was recorded in tumour. Total peroxidase activity was 30–50 times higher in tumour and teratoma than in plantlets. The hypothesis that teratoma tissue with shoots is more similar to plantlets than to unorganised tumour has not been confirmed neither for glucosinolate profiles nor for peroxidase activity, but only for myrosinase activity., Uspoređen je sastav glukozinolata iz in vitro uzgajanih biljčica, tumora i teratoma hrena (Armoracia lapathifolia Gilib.). Biljčice su sadržavale kudikamo više ukupnih glukozinolata nego tumor i teratom. Po količini je prevladavao alifatski glukozinolat. Biljčice su sadržavale još manju količinu aromatskog glukozinolata, glukonasturtina te indolske spojeve: glukobrasicin, 4-metilglukobrasicin i 4-hidroksiglukobrasicin. U tumorskom i teratomskom tkivu otkriveni su samo ti indolni glukozinolati. Aktivnost enzima mirozinaze (β-tioglukozidaza) bila je veća u biljčicama nego u teratomu, a u tumoru nije utvrđena. Ukupna peroksidazna aktivnost bila je 30-50 puta veća u tumoru i teratomu nego u biljčicama. Pretpostavka da će teratomsko tkivo koje sadrži izdanke biti sličnije biljčici nego neorganiziranom tumoru nije se potvrdila ni u profilu glukozinolata, niti peroksidaznoj aktivnosti, nego samo u mirozinaznoj aktivnosti.

Artificial environmental conditions in tissue culture, such as elevated relative humidity and rich nutrient medium, can influence and modify tissue growth and induce spontaneous changes from characteristic organization pattern to unorganized callus. As succulent plants with crassulacean acid metabolism, cacti are particularly susceptible to this altered growth environment. Glycosylated proteins of Mammillaria gracillis tissues cultivated in vitro, separated by SDS-PAGE, were detected with Con A after the transfer of proteins onto the nitrocellulose membrane. The glycan components were further characterized by affinity blotting with different lectins (GNA, DSA, PNA, and RCA(120)). The results revealed significant differences in glycoprotein pattern among the investigated cactus tissues (shoot, callus, hyperhydric regenerant, and tumor). To test whether the N-glycosylation of the same protein can vary in different developmental stages of cactus tissue, the N-glycans were analyzed by MALDI-TOF MS after in-gel deglycosylation of the excised 38-kDa protein band. Paucimannosidic-type N-glycans were detected in oligosaccharide mixtures from shoot and callus, while the hyperhydric regenerant and tumor shared glycans of complex type. The hybrid oligosaccharide structures were found only in tumor tissue. These results indicate that the adaptation of plant cells to artificial environment in tissue culture is reflected in N-glycosylation, and structures of N-linked glycans vary with different developmental stages of Mammillaria gracillis tissues.

Plants sense carbohydrates and transduce a signal which changes gene expression and the activities of many enzymes. The relationship between changes in gene expression by carbohydrates and the developmental state of the cells is still poorly understood. To gain more knowledge about this relationship, we have analyzed three sugar beet (Beta vulgaris L.) in vitro cell lines residing on distinct differentiation states. Cell suspensions were initiated and cells were incubated for 72h in the medium with sucrose as a control, or treated during the same period with glucose or 3-O-methylglucose (3OMG). Cellular and extracellular proteins, separated electrophoretically, showed that sugar-induced protein expression was cell line-specific. More differences were visible in extracellular and in glycoprotein than on cellular protein patterns. The 3OMG downregulated while glucose upregulated cellular glycoproteins. In the case of extracellular proteins, glucose and 3OMG were equally effective in both downregulation and upregulation of protein expression. Sialic acid was indicated as a glycan conjugate in sugar beet. Carbohydrate-induced gene expression was related to the developmental state of cells.

Transgenic plants have recently emerged as a promising system for low-cost and large-scale production of therapeutic proteins. Despite many advantages plants offer as hosts for expression of recombinant proteins, there are certain limitations caused by major structural differences between plant and mammalian N-linked glycans. Thus, efforts are being made in order to eliminate, or at least minimize, plant-specific N-glycosylation as well as to supplement the plant endogenous system with human glycosyltransferases to obtain »humanized« non-immunogenic N-glycans on plant-made pharmaceuticals aimed for human therapy., Nedavno je dokazano da su transgene biljke dobar sustav za jeftinu i obilnu proizvodnju terapeutskih proteina. Usprkos mnogim prednostima biljaka kao domaćina za ekspresiju rekombinantnih proteina, postoje određena ograničenja uzrokovana velikim razlikama u strukturi N-glikana biljaka i sisavaca. Stoga se pokušava ukloniti ili barem smanjiti N-glikozilacija specifična za biljke i proizvesti neimunogene N-glikane iz biljnih lijekova dodatkom humane glikoziltransferaze endogenom biljnom sustavu za primjenu u medicini.

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.

Karakteristika biljnog razvoja je izuzetna plastičnost koja je uvjetovana vanjskim i unutarnjim signalima. Ugljikohidrati su važni čimbenici biljnog razvoja. Biljke mogu osjetiti ugljikohidrate kao disaharide ili monosaharide. Osjet monosaharida može biti ili ovisan ili neovisan o heksokinazi. Nastali signal se prenosi do jezgre gdje utječe na ekspresiju gena. Da li osjet šećera i prijenos signala ovisi o diferencijacijskom stupnju stanice nije poznato. Da bi testirali tu hipotezu, analizirali smo tri stanične linije šećerne repe koje se nalaze na različitim stupnjevima diferencijacije: N – diferencirana, bez morfogeneze ; HO – diferencirana i morfogenetska, i HNO – dediferencirana. Stanične suspenzije dobivene iz kalusa tretirali smo s raznim šećerima i analozima: glukoza, saharoza, 3-O-metilglukoza, 2-deoksiglukoza, manoza i manitol. Pratili smo odgovor nekoliko enzima: invertaze, fenilalanin-amonij lijaze (PAL), i peroksidaze. Rezultati pokazuju da su invertaza stanične stijenke i PAL pogodni za testiranje osjeta šećera u staničnim linijama. Aktivnost tih enzima povisila se u stanicama N i HNO tretiranih s glukozom i saharozom, ali ne i s analozima šećera. Slična aktivacija nije pronađena u stanicama HO. Da bi testirali put prijenosa signala šećera, stanice smo tretirali s inhibitorima proteinskih fosfataza i kinaza te mjerili aktivnost invertaze i PAL. Nije nađena nikakva razlika u odgovoru. U konačnici, podatci govore da postoji veza između stupnja diferencijacije stanice šećerne repe i osjeta šećera, dok je put prijenosa signala šećera u sve tri stanične linije istovjetan.

N-glycans detached from the 42 kDa cellular glycoprotein of in vitro propagated cactus (Mammillaria gracillis Pfeiff.) shoots, callus, hyperhydric regenerants and TW tumour were analyzed by mass spectrometry after in-gel deglycosylation of excised protein bands. Mixtures of N-glycans were shown by the electrospray MS analysis to be highly complex and heterogeneous. Only a low amount of high-mannose N-glycans was present, while the majority of structures were of the complex-type N-glycans. A single N-glycan structure was observed in the shoot sample, which appeared to be in good correlation with previous results. Similarities of assigned structures were revealed in the callus and tumour tissue while the highest number of different oligosaccharide structures was found in the hyperhydric regenerant. The presence of sialic acid as a constituent of N-glycans was indicated by lectin staining, ion exchange chromatography and molecular mapping by mass spectrometry, and could be correlated with the differentiation status of the tissues. Results obtained in this study indicate that different morphological levels can be correlated with the N-glycan composition of cellular glycoproteins., N-glikani, oslobođeni sa staničnoga glikoproteina od 42 kDa iz izdanka, kalusa, hiperhidriranoga regeneranta i tumora TW uzgojenih u kulturi tkiva kaktusa Mammillaria gracillis Pfeiff. analizirani su masenom spektrometrijom nakon deglikozilacije u gelu. Analiza masenom spektrometrijom ESI pokazala je kako su smjese oslobođ enih oligosaharida vrlo složene i heterogene, a većina struktura pripadala je kompleksnome tipu N-glikana. U uzorku izdanka opažena je samo jedna struktura, {to je u skladu s prija{njim rezultatima. Kalusno i tumorsko tkivo pokazalo je mnogo sličnosti u pripisanim strukturama dok je najveći broj različitih oligosaharida prona- đen u hiperhidriranome regenerantu. Na prisutnost sijalinske kiseline ukazala je inkubacija s lektinima, kromatografija ionske izmjene te masena spektrometrija. Rezultati dobiveni u ovim istraživanjima pokazuju da se stupanj organiziranosti tkiva može povezati sa sastavom N-glikana staničnih glikoproteina.

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.

As plants with Crassulacean Acid Metabolism (CAM), cacti are highly affected by artificial environmental conditions in tissue culture. Plants of Mammillaria gracillis Pfeiff. (Cactaceae) propagated in vitro produced callus spontaneously. This habituated callus regenerated normal and hyperhydric shoots without the addition of growth regulators. In order to compare habituated callus with the tumorous one, cactus cells were transformed with two strains of Agrobacterium tumefaciens: the wild strain B6S3 (tumour line TW) and the rooty mutant GV3101 (tumour line TR). Gene expression in cactus plants, habituated callus, regenerated shoots and two tumour lines was analysed at the level of cellular and extracellular protein and glycoprotein profiles. Proteins were separated by SDS-polyacrylamide gel electrophoresis and 2-D PAGE electrophoresis and silver stained. Concavalin A-peroxidase staining detected glycoproteins with D-manose in their glycan component on protein blots. Developmentally specific protein patterns of Mammillaria gracillis tissue lines were detected. The 2-D PAGE electrophoresis revealed some tissue specific protein groups. The cellular glycoprotein of 42 kDa detected by ConA was highly expressed in undifferentiated tissues (habituated callus, TW and TR tumours) and in hyperhydric regenerants. Tumours produced extracellular proteins of 33, 23 and 22 kDa. The N glycosylation of cellular and extracellular proteins was related to specific developmental stage of cactus tissue.

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.

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.

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.

Potato (Solarium tuberosum L. cv. Desirée) tuber tissue was infected with Agrobacterium tumefaciens, strains: B6S3 (wild type), pGV2255 (rooty mutant), pGV2215 (shooty mutant) and with Agrobacterium rhizogenes (8196). Successful tumor induction was achieved by B6S3 (unorganised tumors) and by pGV2255 (rooty tumors). Transformed tuber cells were smaller than normal ones and free from starch. Transformation process caused a significant increase in peroxidase activity which can be taken as a tumorigenetic marker. Electrophoretic protein pattern showed that tumor cells stopped producing a polypeptide of about 40 kd, which was a storage protein dominant in normal tissue., Tkivo gomolja krumpira inficirale smo bakterijom Agrobacterium tumefaciens (sojevi: B6S3, pGV2215, pGV2255) i A. rhizogenes (8196). Uspješnu tumorsku transformaciju postigle smo divljim sojem (B6S3) i mutiranim (pGV2255) koji izaziva tumore s korijenom. Svjetlosnim mikroskopom mogle smo razlikovati normalne stanice od tumorskih. Tumorska transformacija odrazila se u porastu peroksidazne aktivnosti te stoga, ovaj enzim može poslužiti kao pokazatelj transformacijskih procesa. Elektroforetskim razdvajanjem topivih proteina u SDS-poliakrilamidnom gelu utvrdile smo da tumorske stanice prestaju sintetizirati proteinsku prugu na položaju koji odgovara molekularnoj težini od oko 40 kd.

Kod nekoliko kalusnih linija bundeve određena je aktivnost perok- sidaze i dobivena elektroforetska slika njenih izoenzima. Najviša aktivnost enzima utvrđena je kod kalusnnih linija DE i NA3, koje rastu uz dodatak regulatora rastenja i pokazuju visoku sposobnost zametanja embrioida. Kod prilagođenog tkiva (linija Ž5b/T) znatno je niža aktivnost peroksidaze, međutim pojavi embrioida prethodi porast aktivnosti enzima. Kalusne linije razlikuju se međusobno u elektroforetskoj slici izoenzima. Na tu sliku utječu regulatori rastenja, međutim ona je također odraz određenog razvojnog stanja u kulturi.

The development of adventitious buds in Black Pine (Pinus nigra Arn). embryo culture was studied from its beginning to its end, i. e. from the 4th to the 21st day of culturing. The formation of meristemoids brought up in about 12, of primordial buds 16 and of leaf primordia in 19 days. The adventitious buds developed in 21 days and then started to grow. During the culturing of Black Pine embryos the total activity of peroxydases was observed. From the 12th to the 19th day a large increase in peroxidase activity and a characteristic anodic separation pattern of two isoenzymes were stated., Razvitak adventivnih pupova u kulturi embrija crnog bora praćen je histološki i na temelju promjena u aktivnosti i elektroforetskoj slici izoproksidaza. Histološke analize pokazale su da su se adventivni pupovi počeli razvijati oko 4. dana, a njihovo formiranje je bilo okončano oko 21. dana trajanja kulture. Vremenski slijed četiriju sekvencija na koje je razvitak adventivnih pupova bio raščlanjen izgledao je ovako: nastajanje maristemoida bilo je završeno oko 12. dana, primordijalnih pupova oko 16. dana, a primordijalnih listova oko 19. dana nakon čega su rasli sve do kraja kulture. Praćenjem promjena u aktivnosti i elektroforetskoj slici izoperoksidaza utvrđeno je da je ukupna aktivnost peroksidaza u embrijima crnog bora tijekom njihove kulture rasla tako da je između 12. i 19. dana došlo do naglog porasta aktivnosti. Slika izoperoksidaza dobivena anodnim razdvajanjem pokazala je karakteristično razdvajanje na dva izoenzima također između 12. i 19. dana.

The effect of atrazine and of two newly synthesized derivatives of ditriazinil sulphides (4-chloro-4’,6-bis(ethylamino)-6’-isopropylamino-di- (s-triazinyl)-sulphide = CTS and 4-chloro-4’,6-bis(isopropylamino)-6’- ethylamino-di-(s-triazinyl)-sulphide = CPS) on the development and changes of fresh and dry weight of Lemna gibba L. and Spirodela polyr- rhiza L. has been investigated. It has been found that CTS considerably inhibits the growth and the increase of fresh and dry weight of the plants. Its inhibitory effect is, at least in the case of L. gibba stronger than the effect of atrazine (Figs. 1—8.). The results prove that with a higher concentration of the investigated active substances the increase in the number of plants is reduced while the morphological abnormalities become more and more frequent (Figs. 13.—16.). Although the chemical structure of CPS is very similar to CTS its inhibitory effects is much weaker than that of CTS (Figs. 9.—12).