Sugar Beet Cell Research Articles

Nucleic acid and protein elimination during the sugar manufacturing process of conventional and transgenic sugar beets

The fate of cellular DNA during the standard purification steps of the sugar manufacturing process from conventional and transgenic sugar beets was determined. Indigenous nucleases of sugar beet cells were found to be active during the first extraction step (raw juice production) which was carried out at 70°C. This and the consecutive steps of the manufacturing process were validated in terms of DNA degradation by competitive PCR of added external DNA. Each step of the process proved to be very efficient in the removal of nucleic acids. Taken together, the purification steps have the potential to reduce the amount of DNA by a factor of >10 14, exceeding by far the total amount of DNA present in sugar beets. Furthermore, the gene products of the transgenes neomycin phosphotransferase and BNYVV (rhizomania virus) coat protein CP21 were shown to be removed during the purification steps, so that they could not be detected in the resulting white sugar. Thus, sugar obtained from conventional and transgenic beets is indistinguishable or substantially equivalent with respect to purity.

Induction of cell division-related genes in quiescent (G0 ) sugar beet cells.

Sugar beet cells maintained in the stationary phase of the batch culture cycle for 2 or more days have been shown to exhibit many of the characteristics of quiescent (G0 ) cells. When such cells were subcultured into fresh medium they progressed through a period of DNA synthesis to a highly synchronised first division, 6 days after subculture. The onset of DNA synthesis and cell division were each delayed by 2 days relative to the timing of the events when the cells were subcultured immediately before entry into the stationary phase. The regulation of gene expression during this extended transition from the G0 phase back to the cell division cycle was investigated. The cell division cycle-related genes Bvcdc2, Betvu;CycA2, Arath;CycB1;1 histone H4 and Bvcrk1 (a novel cdc2-like gene) showed widely differing patterns of expression. Bvcdc2 transcripts were present at low levels in quiescent cells whereas crk1, cyclin and histone transcripts were not detectable. Expression of both Bvcrk1 and Betvu; CycA2 was induced within 1 h after subculture into fresh medium, whereas histone H4 gene expression was not detectable for 24 h and showed a marked increase between 24 and 48 h. B-type cyclin transcripts were not detectable until more than 48 h after subculture. The addition of either sucrose or MS macronutrients to quiescent sugar beet cells was not sufficient to re-initiate cell division but both medium components were able to stimulate the expression of the two 'early' genes (Betvu;CycA2 and Bvcrk1) within 6 h. Furthermore, although the sugar beet cells were habituated, i.e. they were routinely grown without added plant growth regulators, treatment of quiescent cells with IAA and kinetin also induced expression of Betvu;CycA2 and Bvcrk1 without subsequent cell division.

Biotechnological advances of electroporation of grapevine and sugar beet cells

The aim of this work to characterise factors influencing of the CAT and GUS reporter genes expression using a rectangular pulse generating system, and the interactions between the electrical parameters applied and the physiological state of the electroporated grapevine and sugar beet suspension protoplasts. These results are shown by use of both a rectangular pulse generating system and a more commonly used capacitor discharge system, which delivers pulses of exponentially decaying voltages.

Mechanical behavior of sheets prepared from sugar beet cellulose microfibrils

The mechanical behavior of films cast from sugar beet cellulose microfibrils was investigated through tensile tests. The obtaining of these microfibrils by chemical and mechanical treatments from the raw beet pulp is described. Depending on their purification level, individualization state, and moisture content, differences in tensile modulus are observed. It is found that pectins act as a binder between the cellulose microfibrils, which tends to increase the Young's modulus in dry atmosphere and to decrease it in moist conditions. The extraction of the cellulose microfibrils from the sugar beet cell wall and the obtainment of microfibril suspensions with partial individualization of the microfibrils by a mechanical treatment lead to the formation of a network of cellulose microfibrils within the film, which in turn increases the tensile modulus. Furthermore, the effect of the remaining pectins is compared with the effect of pectins previously removed and added to completely purified cellulosic microfibrils. As expected, once removed and so partly degraded, those pectins have nearly no influence on the mechanical properties. © 1997 John Wiley & Sons, Inc. J Appl Polym Sci 64: 1185–1194, 1997

A boron-rhamnogalacturonan-II complex from bamboo shoot cell walls

A boron-polysaccharide complex was isolated from a Driselase digest of bamboo ( Phyllostachys edulis) shoot cell walls by successive DEAE Sepharose FF, Bio-Gel P-10 and Mono Q HR 5/5 chromatography. The complex contained 0.15% boron (w/w). The glycosyl residue and linkage composition analyses of the polysaccharide moiety of the complex identified the polysaccharide as a rhamnogalacturonan-II (RG-II), a structurally complex pectic polysaccharide present in the primary cell walls of all higher plants. 11B NMR spectroscopy showed that boron was present as a tetrahedral borate-diol diester. Removal of boron from the complex decreased the M r by half without any loss of glycosyl residues, suggesting that boron cross-links two RG-II molecules. The boron-RG-II complex from bamboo (a monocot) shoot cell walls has almost the same structure as that of sugar beet (a dicot) cell walls. The results demonstrate that the structure of boron-RG-II complex is very similar in dicots and monocots.

Polyamine metabolism and gene regulation during the transition of autonomous sugar beet cells in suspension culture from quiescence to division

Sugar beet cells grown in batch suspension culture have been used to study the regulation of polyamine levels during the transition from a quiescent to a proliferating state. The quiescent state was achieved by maintenance of the phytohormone autonomous cells in the stationary phase of the batch culture cycle. After subculture into fresh medium there was an increase in DNA synthesis which was accompanied by a dramatic increase in cellular polyamine levels. The levels of both free and bound cellular putrescine and spermidine within the cells reached a peak before the onset of the first synchronous division. The levels of putrescine, spermidine and to some extent spermine in the culture medium also increased dramatically shortly after subculture. The increase in polyamines was preceded by a rapid but transient increase in omithine decarboxylase (EC 4.1.1.17) and S‐adenosylmethionine decarboxylase (EC 4.1.1.50). Arginine decarboxylase (EC 4.1.1.19) and S‐adenosylmethionine synthetase (EC 2.5.1.6) activity did not show the same pattern of cell division‐related variation. Inhibition of S‐adenosylmethionine biosynthesis with methylglyoxal bis‐(guanylhydra‐zone) (MGBG) reduced cell division in the suspension culture. Inhibitors of ornithine decarboxylase and arginine decarboxylase individually had little effect on cell division, but in combination led to a reduction in cell division. Addition of polyamines and their precursors to cells in the stationary phase of a batch culture cycle led to the induction of expression of a mitotic cyclin sequence (BvcycII).

Polyamine metabolism and gene regulation during the transition of autonomous sugar beet cells in suspension culture from quiescence to division

Polyamine metabolism and gene regulation during the transition of autonomous sugar beet cells in suspension culture from quiescence to division

Isolation and characterization of a boron-rhamnogalacturonan-II complex from cell walls of sugar beet pulp

A boron (B)-polysaccharide complex was isolated from a Driselase digest of sugar beet ( Beta vulgaris L.) cell walls by ion-exchange and gel-permeation chromatography. The complex contained 0.12% B (w/w). The polysaccharide moiety contained 2- O-methylfucose, rhamnose, fucose, 2- O-methylxylose, arabinose, apiose, galactose, aceric acid, galacturonic, and glucuronic acids residues, and thiobarbituric acid-assay positive sugars, presumably 3-deoxy- d-manno-2-octulosonic acid (Kdo) and 3-deoxy- d- lyxo-2-heptulosaric acid (Dha). Methylation analysis, together with glycosyl composition analysis, showed that the polysaccharide was a typical rhamnogalacturonan-II (RG-II), a structurally complex pectic polysaccharide present in the primary cell walls of plants. 11B NMR spectroscopy showed that the B was present as a tetrahedral borate-diol diester. Approximately 70% of B was released by treating the B—RG-II complex at pH 4.8 at 40 °C.

Malondialdehyde cannot be related to lipoperoxidation in habituated sugarbeet plant cells

Malondialdehyde (TBARS: thiobarbituric acid reactive substances) has been investigated in non-organogenic and organogenic habituated and normal sugarbeet cell lines and compared with lipid hydroperoxides, conjugated diene formation and lipoxygenase activity in order to estimate the reality of lipid peroxidation processes which have been postulated in the habituated non-organogenic cell line. The results presented here exhibit a strong discrepancy between TBARS and the other indices tested. Neither hydroperoxide index, nor conjugated dienes, nor lipoxygenase activity could confirm that the non-organogenic habituated cells studied were submitted to permanent stress due to free radical attacks. Moreover, these results underlined that the use of TBARS to estimate lipid peroxidation must be considered with extreme caution in plants and can lead to misinterpretations.

Hydrolysis of glucuronide‐based substrates mediated by tungsten, Cu2+, Fe2+, and Zn2+

A variety of metal microprojectiles are currently used for carrying foreign DNA into living cells via particle‐acceleration techniques. While developing a microprojectile‐mediated protocol for transforming cells of sugarbeet (Beta vulgaris L.), formation of a blue precipitate was observed with the indigoqenic substrate 5‐bromo‐4‐chloro‐3‐indolyl‐β‐D‐glucuronic acid (X‐gluc) in the absence of gusA DNA encoding β‐D‐glucuronidase (GUS). Tungsten microcarriers, but not gold or silicon carbide, proved capable of catalyzing the cleavage of the glucuronide residue from three histochemical substrates evaluated: X‐gluc, salmon X‐gluc and magenta X‐gluc. Indigo‐stained sugarbeet cells were observed following bombardment with tungsten in the absence of DNA. Addition of oxidative catalysts to tungsten microcarriers during substrate incubation had no apparent effect on the metal‐mediated catalysis. Treatment of microcarriers with Proteinase K and heat ruled out the presence of enzymes. Microbiological evaluation indicated the absence of contaminating microbes. Similarly, metal‐catalyzed hydrolysis of the fluorogenic substrate 4‐methylumbelliferyl‐β‐D‐glucuronic acid (4‐MUG) was observed in the presence of tungsten spheres but not with gold or silicon carbide particles. With this substrate, hydrolysis also occurred with millimolar concentrations of Cu2+, Fe2+ and Zn2+ ions. Consequently, careful monitoring of DNA‐minus controls and avoidance of millimolar concentrations of Cu2+, Fe2+ and Zn2+ ions are recommended in microprojectile bombardment experiments where transient assays for gusA expression are performed.

Habituation in sugarbeet plant cells: Permanent stress or antioxidant adaptative strategy?

Habituation is one of the four neoplastic diseases of plants and occurs spontaneously in plant cell cultures. To date, and even if an epigenetic origin has been previously postulated, the fundamental concept that underlies this neoplastic state remains obscure. Recently, a permanent stress hypothesis has been proposed, using habituated nonorganogenic (HNO) sugarbeet cell line (Beta vulgaris L.altissima) as a model. According to this proposal, the low catalase and peroxidase activities were supposed to be responsible for H2O2 accumulation. A supposed lipoxygenase activation would generate LOO° radicals. OH°, produced by the Fenton reaction would be responsible for a lipoperoxidation process, leading to malondialdehyde (MDA) accumulation. In this paper, the elements of this hypothesis have been examined using data previously obtained by several teams, and the permanent stress idea appears less sustainable. Several properties of the habituated nonorganogenic sugarbeet- and some other habituated-cell lines have been described. A more realistic concept emerging from this analysis is that habituated cells exhibit efficient scavenging properties (antioxidant and antilipoperoxidant) against deleterious free radicals produced during cell culture. This thesis is developed in this article.

Purification and properties of NAD(P)H: (quinone-acceptor) oxidoreductase of sugarbeet cells.

NAD(P)H:(quinone-acceptor) oxidoreductase [NAD(P)H-QR], a plant cytosolic protein, was purified from cultured sugarbeet cells by a combination of ammonium sulfate fractionation, FPLC Superdex 200 gel filtration, Q-Sepharose anion-exchange chromatography, and a final Blue Sepharose CL-6B affinity chromatography with an NADPH gradient. The subunit molecular mass is 24 kDa and the active protein (94 kDa) is a tetramer. The isoelectric point is 4.9. The enzyme was characterized by ping-pong kinetics and extremely elevated catalytic capacity. It prefers NADPH over NADH as electron donor (kcat/Km ratios of 1.7 x 10(8) M-1 S-1 and 8.3 x 10(7) M-1 S-1 for NADPH and NADH, respectively, with benzoquinone as electron acceptor). The acridone derivative 7-iodo-acridone-4-carboxylic acid is an efficient inhibitor (I0.5 = 5 x 10(-5) M), dicumarol is weakly inhibitory. The best acceptor substances are hydrophilic, short-chain quinones such as ubiquinone-0 (Q-0), benzoquinone and menadione, followed by duroquinone and ferricyanide, whereas hydrophobic quinones, cytochrome c and oxygen are reduced at negligible rates at best. Quinone acceptors are reduced by a two-electron reaction with no apparent release of free semiquinonic intermediates. This and the above properties suggest some relationship of NAD(P)H-QR to DT-diaphorase, an animal flavoprotein which, however, has distinct structural properties and is strongly inhibited by dicumarol. It is proposed that NAD(P)H-QR by scavenging unreduced quinones and making them prone to conjugation may act in plant tissues as a functional equivalent of DT-diaphorase.

The effect of calcium chelators on microsomal pyridine nucleotide-linked dehydrogenases of sugarbeet cells

Cell suspension cultures of Beta vulgaris L., treated with calcium chelators or untreated, were used to characterize pyridine nucleotide-dependent diaphorases of microsomes. The microsomal activity of NADH-dependent duroquinone reductase from cultures treated with 10 mM Na 2 EGTA for 24 h increased by a factor of 1.8 with respect to control microsomes, and was mainly associated with particles of d=1.17 g ml -1 . NADPH-duroquinone reductase and NADH-ferricyanide reductase activities showed smaller increases. Bacterial protein-lipopolysaccharide complexes (prLPS) also promoted the increase of microsomal diaphorases; CaEGTA was ineffective. EGTA effects on enzymes of supernatant and mitochondria were negligible, although Na 2 EGTA treatment induced cell aggragation and strong acidification of the medium

Calcium Retrieval from Vacuolar Pools (Characterization of a Vacuolar Calcium Channel).

Voltage patch-clamp experiments at the whole-vacuole and single-channel levels were employed to study the retrieval of Ca2+ from vacuoles into the cytoplasm in sugar beet cell (Beta vulgaris L.) suspension cultures. Channels allowing the movement of Ca2+ out of the vacuole were identified at physiological conditions of pH, vacuolar membrane potential, and vacuole/cytoplasm Ca2+ concentrations. The operation of the channel was voltage dependent and inositol-1,4,5-triphosphate insensitive and displayed high selectivity for Ca2+ ions. These channels bear similarities to the dihydropyridine-sensitive L-type Ca2+ channels from animal cells. Bay K-8644, an agonist, increased the frequency of channel openings, whereas nifedipine, an antagonist, reduced the channel activity. Both effects were elicited only from the vacuolar side of the channel. Channel activities were also inhibited by verapamil, La3+, and cytoplasmic Ca2+ concentrations higher than 1 x 10-6 M. The modulation of the channel currents by cytoplasmic Ca2+ would suggest the role of these channels in triggering the initiation of signal transduction processes in plant cells.

Rearrangements in sugar beet mitochondrial DNA induced by cell suspension, callus cultures and regeneration

Structural alterations in mitochondrial DNAs (mtDNAs) from a plant of a sterile sugar beet line, callus derived from it, suspension-cultured cells and plants regenerated from the callus were studied. BamHI restriction analysis revealed that structural alterations between the mtDNAs of the callus and the control plant had occurred. Multiple rearrangements were also demonstrated in the mtDNA from the suspension culture, of which some were similar to those appearing in the callus, and others had arisen de novo. Rearrangements were also identified by means of blot hybridization of BamHI-digested mtDNA from suspension-cultured cells with the genes encoding subunit II of cytochrome oxidase (cox II) and subunit 1 of NADH-dehydrogenase (Nd1). No alterations were observed in the mitochondrial genome of the callus and regenerants. The location of the genes for the α-subunit of F1-ATPase (atpA) and apocytochrome b (cob) in the mtDNA remained unchanged.Our salient finding was of a plant with an altered mitochondrial genome as judged by EcoRI and BamHI restriction analysis. This exceptional plant had retained the sterile phenotype like all of the other regenerants and the parent. The set of plasmid-like molecules of mtDNA remained the same as that in the control plant and in all of the regenerants, callus and suspension-cultured cells. The only type of plasmid-like molecule found in all of the DNAs was the 1.6-kbp minicircle, which is a feature of sterile cytoplasms. These structural changes in mtDNA were obviously a consequence of somaclonal variation during the in vitro cultivation of the sugar beet cells.

Structure and properties of apple and sugar-beet pectins extracted by chelating agents

Pectins were extracted from apple or sugar-beet cell walls (alcohol-insoluble solids) at pH 4.5 and 20°C and at pH 6.5 and 80°C in acetate and phosphate buffer solutions, respectively, and in the same buffers containing cyclohexanediaminetetraacetic acid or ethylenediaminetetraacetic acid. The yields were very different between the two pH and temperature conditions, but variations with the nature of the extractant were small at pH 4.5 and 20°C, and no differences were observed at pH 6.5 and 80°C. The pectins extracted at pH 6.5 and 80°C were richer in neutral sugars and acetic acid. Degradation of the pectins extracted at high pH and temperature was shown by the decrease of the intrinsic viscosity and by the shift of the uronic acid peak toward the total volume in gel-filtration chromatography. These pectins contained a high molecular weight population rich in neutral sugars and a low molecular weight acid population, in contrast to the pectins extracted at low temperature and pH, for which neutral and acidic sugars had similar elution profiles. At pH 6.5 and 80°C, rather extensive degradation of the pectins occurred. Calcium-binding experiments (potentiometry with specific electrode and conductimetry) indicated a statistical distribution of free carboxyl groups. These results do not support the hypothesis that chelating-agent extractable pectins are held in the cell wall by calcium cross-links involving the formation of “egg-boxes”.

A pectin Lyase Inhibitor Protein from Cell Walls of Sugar Beet

A protein from sugar beet roots inhibited pectin lyase (PNL) that was isolated from Rhizoctonia solani. The PNL inhibitor protein (PNLIP) was extracted from healthy sugar beet cell walls and partially purified by ammonium sulfate fractionation, cation exchange, and gel permeation chromatography. Electrophoresis in polyacrylamide with sodium dodecyl sulfate showed a single major band of 57.5 kDa. Isoelectric focusing gave a major band at pI 9.9. Kinetic analysis indicated that the inhibition was uncompetitive (coupling). Pectin lyase inhibitory activity was greater in cell wall extracts from rotted and adjacent tissue than in healthy tissue []

Characterization of Vacuolar Malate and K+ Channels under Physiological Conditions

Patch-clamp techniques were employed to study the electrical properties of vacuoles from sugar beet (Beta vulgaris) cell suspensions at physiological concentrations of cytoplasmic Ca(2+). Vacuoles exposed to K(+) malate revealed the activation of instantaneous and time-dependent outward currents by positive membrane potentials. Negative potentials induced only instantaneous inward currents. The time-dependent outward currents were 10 times more selective for malate than for K(+) and were completely blocked by zinc. Vacuoles exposed to KCl developed instantaneous currents when polarized to positive or negative membrane potentials. The time-dependent outward channels could serve as the route for the movement of malate into the vacuole, whereas K(+) could move through the time-independent inward and outward channels.

Construction and characterisation of a yeast artificial chromosome library containing two haploid Beta vulgaris L. genome equivalents

We have constructed a yeast artificial chromosome (YAC) library using high-molecular-weight DNA prepared from agarose-embedded protoplasts of a sugar beet ( Beta vulgaris L.) cell suspension line, Ar +. This library contains 15 000 clones with an average insert size of 140 kb. Based on a sugar beet haploid genome size of 1.1 × 10 3 Mb it should represent approximately two haploid genome equivalents. The library is organised as an ordered array in duplicate microtitre plates. High-density filters, each containing 864 YAC clones, have been screened m colony hybridisation experiments. In this way, sugar beet YAC clones have been identified containing chloroplast DNA, mitochondrial DNA and two satellite DNAs. Ten pools of DNA from 1500 individual YAC clones have also been prepared for rapid PCR screening of the library. Using this approach, in combination with colony hybridisation, we have been able to isolate sugar beet YAC clones containing a transcribed low-copy-number gene.

Wall-bound α-glucosidase of suspension-cultured sugar-beet cells

Wall-bound α-glucosidase showed similar properties to other α-glucosidases produced by suspension-cultured sugar-beet cells. The wall-bound enzyme could not be solubilized from wall pellets by high salt concentrations, detergents or a combination of 8 M urea and 0.1 M sodium sulphite. A polysaccharide susceptible to α-glucosidase digestion was also contained in the wall pellets. β-Amylase and glucoamylase released, respectively, 20 and 49% of the carbohydrate.