Sugar Beet Cell Research Articles

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 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.

Влияние ферментативной обработки на качество технологических соков при производстве сахара из бактериально инфицированной сахарной свеклы

Объектом исследований служили корнеплоды инфицированной слизистым бактериозом сахарной свеклы, клеточный сок и полуфабрикаты свеклосахарного производства. Цель исследований заключалась в установлении закономерностей изменения качества технологических соков при введении ферментного препарата гликозидазного действия в процессе экстрагирования сахарозы из бактериально инфицированной сахарной свеклы. Теоретически обоснованы предпосылки применения гликозидазных ферментных препаратов при переработке бактериально инфицированной сахарной свеклы. На основе физического и математического моделирования процессов экстрагирования сахарозы и известково-углекислотной очистки диффузионного сока выявлены зависимости изменения технологических показателей от дозы введения гликозидазного ферментного препарата и степени инфицированности слизистым бактериозом клеточного сока сахарной свеклы. Показана роль ферментативной обработки в улучшении качества технологических соков, в том числе увеличении содержания сахарозы в очищенном соке в среднем на 2,2%, повышении эффекта очистки диффузионного сока в среднем на 3,5%, снижении мутности очищенного сока – на 20%, что может обеспечить повышение качества и выход белого сахара на 1,1%. Достигнуто требуемое состояние пищевой системы с оптимальными (максимальными) показателями величины коэффициента желательности от 0,63 до 0,99, что соответствует хорошему и очень хорошему уровню в зависимости от степени инфицированности корнеплодов. Дальнейшие исследования будут направлены на обоснование условий рационального применения ферментных препаратов гликозидазного действия в оптимальной комбинации совместно с другими технологическими вспомогательными средствами при экстрагировании сахарозы из бактериально инфицированной сахарной свеклы.

Natural deep eutectic solvents are viable solvents for plant cell culture-assisted stereoselective biocatalysis

We report a plant (Beta vulgaris L. subsp. vulgaris.) cell culture-mediated reduction of prochiral 1-(3,4-dimethylphenyl)ethanone into the chiral alcohol (1R)-1-(3,4-dimethylphenyl)ethanol in natural deep eutectic solvents (NADES). To the best of our knowledge, this is the first report on plant culture cell behaviour during incubation in these solvents, where the novelty is transformation in callus culture. Three different choline chloride-based NADES (aqueous solutions containing 30, 50 and 80 % water [w/w]) containing sugar (glucose) or polyalcohols (glycerol and ethylene glycol) were screened for conversion and enantiomeric excess during the bioreduction. Both the conversion and the enantiomeric excess differed considerably when using different hydrogen bond donors, with the (R)-alcohol configuration predominating in the reactions conducted in most NADES. Changing the water content in NADES also altered the enantioselectivity. Testing the biocompatibility of NADES with sugar beet cell cultures revealed that NADES cause permeabilisation of cell membranes, leading to stress conditions that change plant metabolism. The potential for recycling and reusing plant biomass was tested for sugar beet cell cultures. The results indicate that recycling may be possible after 3–7 days of incubation, but longer incubations lead to too high a toxicity to cellular metabolism.

Perspective Technologies of Isolated Tissue Culture in the Sugar Beet Breeding Process

The methods of isolated tissue cultures allowing production and rapid propagation of the sugar beet plant material with improved or new traits are described in the presented study. It was shown that the proposed original scheme of mass reproduction and in vitro deposition of elite plants—components of highly productive hybrids—can be used for the production of seeds with improved quality. The technology of in vitro selective selections carried out under abiotic stress conditions allows for the production of sugar beet isogenic lines with high resistance to external environment abiotic factors (salinization, drought, soil acidity). The effect of certain factors of in vitro culture on haploid cells of sugar beet ovules allowed production of doubled haploid lines (DH) with high homozygosity level and qualitative seeds of highly productive hybrid components. The developed technologies based on biotechnological methods are a preferred and innovative direction of research in modern science, since the introduction of these developments in the breeding process will contribute to the production of competitive hybrids with a set of desired economic traits.

Plant-mediated asymmetric reduction of 1-(3,4-dimethylphenyl)ethanone

Bioreduction mediated by plants is a highly selective and environmentally friendly approach to synthesise optically active alcohols. Herein the bioreduction of 1-(3,4)-dimethylphenyl)ethanone to the corresponding chiral alcohol 1-(3,4-dimethylphenyl)ethanol has been evaluated using nine different vegetables roots as biocatalysts. The chiral alcohol was prepared in yields ranging from 44.1% to 88.2%, and with enantiomeric excess up to 97.2% for the (S)-enantiomer. Sugar beet was the most promising among the tested vegetables roots. Therefore, sugar beet cell cultures (normal and transformed) were obtained and tested for the reduction as well, yielding the corresponding alcohol in the range from 62.1% to 88.2% yield and with enantiomeric excesses of >99%. Based on these results reported, there are no profound differences in the reductive capacity of undifferentiated cells and organs of the same plant species (sugar beet). Due to overall advantages of using cell cultures, we have highlighted this approach as a promising method for preparation of enantiomerically pure 1-(3,4-dimethylphenyl)ethanol.

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 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.

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.

Transgenic Varieties: Sugarbeet

The development and market introduction of H7-1, the first commercially relevant transgenic sugarbeet, is described. A gene which sequence had been modified to code for a glyphosate-insensitive 5-enolpyrovylshikimate-3-phosphate synthase (EPSPS) was transferred to sugarbeet cells using Agrobacterium-mediated transformation. Plants were regenerated from such cells and were submitted to rigorous testing of their molecular, physiological, agronomic, and ecological properties. Data from these analyses constituted the basis for the legal deregulation of an elite transformation event. As results of this registration process permits for the cultivation of H7-1 have been issued in the USA, Canada, and Japan. For import purposes, food and feed utilization permits are in place in more than 15 countries, including the EU. H7-1 plants were used as starting material for the development of varieties adapted to the respective agronomical requirements of the designated cultivation areas, making extensive use of marker-assisted backcrossing (MABC) as one of the breeding tools. In addition to herbicide tolerance, numerous other transgenic traits are currently under development. Their prospects and technical concepts are briefly discussed.

Identification of Extensin Protein Associated with Sugar Beet Pectin

Several studies have suggested that the emulsification properties associated with pectin obtained from sugar beet (Beta vulgaris) are due to the presence of a protein-pectin complex. Nevertheless, the identity of the protein has remained elusive. Pectin, extracted from sugar beet pulp by microwave-assisted extraction, and a commercial sample were both subjected to protease digestion with trypsin. The resulting peptides were separated from the pectin solution by ultrafiltration using a 3 kDa molecular weight cutoff (MWCO) membrane and analyzed using matrix-assisted laser desorption ionization with tandem time-of-flight mass spectrometry. The partial sequences derived from the mass spectrometry analyses of the resulting tryptic peptides are found to be highly consistent with extensin protein matched from the B. vulgaris Genetic Index database and also correspond to previously reported extensin peptides found in sugar beet cell suspension cultures. Further attempts were made to disassociate the protein from pectin using 1 M NaCl and a 100 kDa MWCO membrane; however, no peptides were observed following trypsin digestion of the permeate solution. This evidence suggests the existence of a complex between the pectin and extensin that is not due to ionic interactions. Trypsin digestion of commercial sugar beet pectin also produced the peptide profile observed with the microwave-assisted extracted pectin sample. Atomic force microscopy established that the number of rod-like elements decreased following protease treatment compared to the untreated sample.

Foliar spray of a cell wall protein fraction from the biocontrol agent Pythium oligandrum induces defence-related genes and increases resistance against Cercospora leaf spot in sugar beet

After a cell wall protein fraction (CWP) of Pythium oligandrum was sprayed on sugar beet leaves, we screened leaves for induced expression of defence-related genes and for resistance against Cercospora leaf spot. In a western blot analysis, the CWP was primarily retained on the surface of leaves without degradation for at least 48 h after spraying. In northern blot analyses, four defence-related genes (β-1, 3-glucanase, acidic class III chitinase, 5-enol-pyruvylshikimate-phosphate synthase and oxalate oxidase-like germin) were expressed more rapidly in CWP-treated leaves compared to control leaves treated with distilled water (DW). When CWP was applied to a suspension of cultured cells of sugar beet, an oxidative burst was observed that did not occur after the DW treatment. In growth chamber trials after inoculation with Cercospora beticola, the severity of Cercospora leaf spot was significantly reduced in CWP-treated plants compared to the DW-treated controls. In a field experiment, CWP treatment was also effective against the disease. CWP did not reduce growth rate of the pathogen in plate tests. The results together suggest that the CWP from P. oligandrum can be retained on the leaf surface and induce expression of disease resistance genes, thereby reducing Cercospora leaf spot on sugar beet.

The differences in cell wall composition in leaves and regenerating protoplasts of Beta vulgaris and Nicotiana tabacum

Cell wall composition in leaves and regenerating leaf-derived protoplasts was studied in Beta vulgaris L. and Nicotiana tabacum L. Several oligosaccharides that characterize arabinogalactan proteins (AGPs) and pectins were visualized in situ by a series of immunocytochemical reactions. The most conspicuous difference between the two species involved the expression of AGP epitopes that bind LM2 and MAC207 in only sugar beet cells; both epitopes being characterized by the presence of glucuronic acid (GlcA). Moreover, B. vulgaris leaves and protoplast-derived cells showed lower content of pectin side-chains bearing (1→4)-β-D-galactose residues as compared to N. tabacum.

Assessment of In Vitro Binding of Isolated Pectic Domains to Cellulose by Adsorption Isotherms, Electron Microscopy, and X-ray Diffraction Methods

Isolated pectic domains representative of the pectic backbone and the neutral sugar side chains were tested for their ability to interact with cellulose in comparison to the well-known binding of xyloglucan. Pectic side chains displayed a significant in vitro binding capacity to cellulose, whereas pectic backbone domains exhibited only slight adsorption to cellulose microfibrils. To support the binding results, electron microscopy and X-ray diffraction were applied. Celluloses from bacteria and sugar beet cell walls were used as substrates for the precipitation of isolated pectic domains or xyloglucan by acetone vapor diffusion. Pectic side chains grew attached to the cellulose surfaces, whereas pectic backbone domains were observed separately from cellulose microfibrils. Xyloglucan seeded with cellulose provoked a decrease of microfibrils entanglement, but no clear cross-links between neighboring microfibrils were observed. These results led to the elucidation of the pectic domains responsible for binding with cellulose microfibrils.

Relationship between DNA methylation and histone acetylation levels, cell redox and cell differentiation states in sugarbeet lines

In order to evaluate the permanent chromatin remodeling in plant allowing their high developmental plasticity, three sugarbeet cell lines (Beta vulgaris L. altissima) originating from the same mother plant and exhibiting graduate states of differentiation were analyzed. Cell differentiation has been estimated by the cell redox state characterized by 36 biochemical parameters as reactive oxygen species steady-state levels, peroxidation product contents and enzymatic or non-enzymatic protective systems. Chromatin remodeling has been estimated by the measurement of levels of DNA methylation, histone acetylation and corresponding enzyme activities that were shown to differ between cell lines. Furthermore, distinct loci related to proteins involved in cell cycle, gene expression regulation and cell redox state were shown by restriction landmark genome scanning or bisulfite sequencing to display differential methylation states in relation to the morphogenic capacity of the lines. DNA methylating, demethylating and/or histone acetylating treatments allowed to generate a collection of sugarbeet cell lines differing by their phenotypes (from organogenic to dedifferentiated), methylcytosine percentages (from 15.0 to 43.5%) and acetylated histone ratios (from 0.37 to 0.52). Correlations between methylcytosine or acetylated histone contents and levels of various parameters (23 or 7, respectively, out of 36) of the cell redox state could be established. These data lead to the identification of biomarkers of sugarbeet morphogenesis in vitro under epigenetic regulation and provide evidence for a connection between plant morphogenesis in vitro, cell redox state and epigenetic mechanisms.

Alkaline extractability of pectic arabinan and galactan and their mobility in sugar beet and potato cell walls

The organisation of sugar beet and potato cell walls was studied using alkaline extractions following a response surface methodology, simultaneously with solid-state 13C NMR spectroscopy. The influence of two extraction parameters: NaOH concentration (0.05, 0.275, 0.5 M) and temperature (40, 65, 90 °C) on the composition (neutral and acidic sugars) of the residues recovered was established. Treatments of increasing harshness progressively washed off non-cellulosic polysaccharides from the cell walls. Alkaline treatments applied to sugar beet cell wall material (SB-CWM) revealed the presence of diverse pectin populations. The existence of distinct pectin populations in potato cell wall material (P-CWM) was less outstanding. Solid-state 13C NMR applied to SB-CWM and P-CWM and residues after treatment by 0.275 M NaOH at 65 °C revealed two fractions of pectic arabinan and galactan side chains. One fraction was highly mobile, whereas the other one displayed restricted mobility.

Sugar beet ( Beta vulgaris) pectins are covalently cross-linked through diferulic bridges in the cell wall

Arabinan and galactan side chains of sugar beet pectins are esterified by ferulic acid residues that can undergo in vivo oxidative reactions to form dehydrodiferulates. After acid and enzymatic degradation of sugar beet cell walls and fractionation of the solubilized products by hydrophobic interaction chromatography, three dehydrodiferulate-rich fractions were isolated. The structural identification of the different compounds present in these fractions was performed by electrospray-ion trap-mass spectrometry (before and after 18O labeling) and high-performance anion-exchange chromatography. Several compounds contained solely Ara (terminal or α-1 → 5-linked-dimer) and dehydrodiferulate. The location of the dehydrodiferulate was assigned in some cases to the O-2 and in others to the O-5 of non-reducing Ara residues. One compound contained Gal (β-1 → 4-linked-dimer), Ara (α-1 → 5-linked-dimer) and dehydrodiferulate. The location of the dehydrodiferulate was unambiguously assigned to the O-2 of the non-reducing Ara residue and O-6 of the non-reducing Gal residue. These results provide direct evidence that pectic arabinans and galactans are covalently cross-linked (intra- or inter-molecularly) through dehydrodiferulates in sugar beet cell walls. Molecular modeling was used to compute and structurally characterize the low energy conformations of the isolated compounds. Interestingly, the conformations of the dehydrodiferulate-bridged arabinan and galactan fragments selected from an energetic criterion, evidenced very nice agreement with the experimental occurrence of the dehydrodiferulated pectins. The present work combines for the first time intensive mass spectrometry data and molecular modeling to give structural relevance of a molecular cohesion between rhamnogalacturonan fragments.

DNA methylating and demethylating treatments modify phenotype and cell wall differentiation state in sugarbeet cell lines

In plants organogenesis, cell differentiation and dedifferentiation are fundamental processes allowing high developmental plasticity. Such plasticity involved epigenetic mechanisms but limited knowledge is available concerning quantitative aspects. Three sugarbeet ( Beta vulgaris L. altissima) cell lines originating from the same mother plant and exhibiting graduate states of morphogenesis were used to assess whether these differences could be related or not to changes in DNA methylation levels. Methylcytosine percentages from 18.3 to 28.8% and distinct levels of DNA methyltransferase (EC 2.1.1.37) activities were shown in the three cell lines. The lowest methylcytosine percentage was associated to organogenesis. In order to test the plasticity of these cell lines, various treatments causing DNA hypo or hypermethylation were performed at different times and concentrations. In this collection of treated lines with ± 10% of methylcytosine percentages, loss of organogenic properties and cell dedifferentiation were observed. As cell wall formation fits well with cell differentiation state, the lignification process was further investigated in treated and untreated lines as a biochemical marker of the phenotypic changes. For example, peroxidase specific activities (EC 1.11.1.7) varied from 0.7 to 0.02 pkat mg –1 of protein in organogenic and dedifferentiated lines, respectively. A negative relationship between peroxidase activities, incorporation of cell wall-bound phenolic compounds as ferulate and sinapate derivatives and methylcytosine percentages was obtained. This is the first biochemical evidence that phenotypic changes in plant cells induced by DNA hypo- or hypermethylating treatments are correlated in a linear relationship to modifications of the cell wall differentiation state.

Isolation of diferulic bridges ester-linked to arabinan in sugar beet cell walls

After degradation of sugar beet cell walls with Driselase® and fractionation of the solubilised products by hydrophobic interaction chromatography, a dehydrodiferuloylated oligoarabinan was isolated. Its structure was assigned to two dimers of (1 → 5)-linked arabinose units esterified by a central 8-O-4′ ferulic dimer. These results provide the first direct evidence that pectic arabinans in sugar beet cell walls may be covalently cross-linked through dehydrodiferulates.

Epiplastome Variation of the Number of Chloroplasts in Stomata Guard Cells of Sugar Beet (Beta vulgaris L.)

In stomata guard cells of sugar beet, variation in the number of chloroplasts was studied in successive generations: (1) hybrid generation; (2) generation yielded by uniparental apozygotic seed reproduction; (3) generation obtained after seed treatment with a colchicine solution; (4) generation obtained after seed treatment with 5-azacytidine. As compared to hybrid generation, uniparental seed reproduction increases the average number of chloroplasts in stomata guard cells (from 13.5 to 15.0) and decreases distribution variance of this trait by a factor of 3 to 4. Colchicine increases both average number of chloroplasts in stomata guard cells (from 13.5 to 18.2) and distribution variance (about twice). 5-Azacytindine reduces the number of chloroplasts in cells (from 15.0 to 12.9) but enhances distribution variance (about 1.5 times). Variation in the number of chromosomes in stomata cells is related to myxoploidy in meristem tissue, on the one hand, and to the rate of cell division, on the other. Uniparental seed reproduction is suggested to enhance the number of organelles per cell due to high myxoploidy in cell populations, which is typical of inbred plants. Colchicine blocks spindle division and sharply increases the level of myxoploidy in cell populations and the number of organelles per cell. 5-Azacytidine hypomethylates chromosome DNA, increases the rate of cell divisions, and reduces the number of organelles per cell. The described changes in the number of chloroplasts are inherited in cell lineage ("cell hereditary memory") and successive sporophyte generations.

Isolation from sugar beet cell walls of arabinan oligosaccharides esterified by two ferulic acid monomers.

Side chains of sugar beet (Beta vulgaris) pectins, which are mainly composed of arabinose (Ara) and galactose (Gal) residues, are esterified by ferulic acid units. Enzymatic hydrolysis of beet cell walls yielded several feruloylated oligosaccharides, which were separated by hydrophobic interaction chromatography. Two new oligomers were isolated in the fraction eluted by 25:75 (v/v) ethanol:water. An arabinotriose and an arabinotetraose esterified by two ferulic acid residues were obtained, and their structure was elucidated by mass spectrometry. It is shown that feruloyl groups are linked to O-5 of Ara residues, in addition to the known O-2 position. This work establishes for the first time, to our knowledge, that two neighboring Ara units may be esterified by two ferulic acid units. This close proximity may have important biochemical implications.

Transformation of sugar beet cell suspension cultures

Summary A sugar beet transformation method was developed using particle bombardment of short-term suspension cultures of a breeding line FC607. Highly embryogenic suspension cultures derived from leaf callus were bombarded with the uidA (gusA) reporter gene under the control of either the osmotin or proteinase inhibitor II gene promoter, and the npt II selectable marker gene. Transient uidA expression was visualized as 500– 4000 blue units per 200 mg of bombarded cells 2 d after bombardment. Stably-transformed calluses were recovered on both kanamycin and paromomycin media. The greatest number of GUS (þ ) calluses was obtained when 50 or 100 mg l 21 of kanamycin was applied 2 d after transformation for 3 –5 wk, followed by either no selection or reduced levels of the antibiotic. PCR analyses of the GUS (þ) callus lines revealed the expected size fragment for uidA and npt II genes. Stable incorporation of the uidA gene into the genome was confirmed by Southern blot analyses. Several transformed embryos were detected by histochemical b-glucuronidase (GUS) staining.