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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.

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.

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.

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.

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.