Enhancement of betanin yield in transformed cells of sugar beet (Beta vulgaris L.)

Betanin belongs to a large family of betacyanin pigments, betalains, and in the food industry it is established as a powerful antioxidant and natural colorant. The main source of betanin is a red beet (Beta vulgaris L.), the yield depending on abiotic and biotic factors in the field. Sugar beet cells, transformed by a wild strain B6S3 of Agrobacterium tumefaciens, strongly produce betanin and could be a stable production source.With the aim of enhancing the yield, cell suspensions were initiated from friable calli. Biomass accumulation, betanin content and yield were monitored over 21 days in relation to changes of sucrose concentration, modifications of minerals in nutrient medium, and the usage of elicitors. Results showed that elevating sucrose levels from 3%to 4%, 5%, or 6%(w/v) in the original medium strongly induced biomass accumulation followed by an increase in betanin yield of up to 250%. Modification of minerals in the medium additionally increased betanin yield up to 20% in a few instances: 40 mg L–1 was recorded at day 10 with 5% and 6% of sucrose. The highest yield at 53 mg L–1 was reached at day 21 on 4% Suc, again with the modified medium. High sucrose concentrations positively affected betanin accumulation only during lag phase of the cell suspension, but afterwards the trend reversed. Calcium and yeast extract were used as abiotic and biotic elicitor, respectively, in the early exponential phase of subculture (day 7). Calcium ions (at 10 fold higher concentration than in the control) failed to increase betanin yield as well as yeast extract at 0.25% (w/v). Yeast extract at 1.25% provoked excretion of betanin at day 4, and cell necrosis at day 7 after elicitation. Taken together, in our system, sucrose affected betanin yield more strongly than medium modifications or elicitors. Yeast extract could be used for reverse- sequestration of betanin where the cells can be used over an extended period

Složena biokemija i biotehnološka proizvodnja betalaina

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

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 GNAand PNAlectin 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

Evaluation of Protein Extraction Methods for Proteomic Analysis of Non-Model Recalcitrant Plant Tissues

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

Sugar-induced changes in cellular and extracellular protein and glycoprotein patterns of sugarbeet cell lines

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

Sugar beet cells’ cellular and extracellular events taking place in response to drought and salinity

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 nutrient medium (PG0). Severe stress altered the intracellular concentrations of most of the 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 affected the expression of cellular and extracellular proteins in sugar beet cells

Sugar-induced changes in cellular and extracellular protein and glycoprotein patterns of sugarbeet cell lines

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

Uloga enzima u metabolizmu ugljikohidrata pri diferencijaciji staničnih linija šećerne repe

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