Habituated Cell Line Research Articles

Phenolic metabolism and molecular mass distribution of polysaccharides in cellulose-deficient maize cells.

As a consequence of the habituation to low levels of dichlobenil (DCB), cultured maize cells presented an altered hemicellulose cell fate with a lower proportion of strongly wall-bound hemicelluloses and an increase in soluble extracellular polymers released into the culture medium. The aim of this study was to investigate the relative molecular mass distributions of polysaccharides as well as phenolic metabolism in cells habituated to low levels of DCB (1.5 μM). Generally, cell wall bound hemicelluloses and sloughed polymers from habituated cells were more homogeneously sized and had a lower weight-average relative molecular mass. In addition, polysaccharides underwent massive cross-linking after being secreted into the cell wall, but this cross-linking was less pronounced in habituated cells than in non-habituated ones. However, when relativized, ferulic acid and p-coumaric acid contents were higher in this habituated cell line. Feasibly, cells habituated to low levels of DCB synthesized molecules with a lower weight-average relative molecular mass, although cross-linked, as a part of their strategy to compensate for the lack of cellulose.

Early cell-wall modifications of maize cell cultures during habituation to dichlobenil

Studies involving the habituation of plant cell cultures to cellulose biosynthesis inhibitors have achieved significant progress as regards understanding the structural plasticity of cell walls. However, since habituation studies have typically used high concentrations of inhibitors and long-term habituation periods, information on initial changes associated with habituation has usually been lost. This study focuses on monitoring and characterizing the short-term habituation process of maize (Zea mays) cell suspensions to dichlobenil (DCB). Cellulose quantification and FTIR spectroscopy of cell walls from 20 cell lines obtained during an incipient DCB-habituation process showed a reduction in cellulose levels which tended to revert depending on the inhibitor concentration and the length of time that cells were in contact with it. Variations in the cellulose content were concomitant with changes in the expression of several ZmCesA genes, mainly involving overexpression of ZmCesA7 and ZmCesA8. In order to explore these changes in more depth, a cell line habituated to 1.5μM DCB was identified as representative of incipient DCB habituation and selected for further analysis. The cells of this habituated cell line grew more slowly and formed larger clusters. Their cell walls were modified, showing a 33% reduction in cellulose content, that was mainly counteracted by an increase in arabinoxylans, which presented increased extractability. This result was confirmed by immunodot assays graphically plotted by heatmaps, since habituated cell walls had a more extensive presence of epitopes for arabinoxylans and xylans, but also for homogalacturonan with a low degree of esterification and for galactan side chains of rhamnogalacturonan I. Furthermore, a partial shift of xyloglucan epitopes toward more easily extractable fractions was found. However, other epitopes, such as these specific for arabinan side chains of rhamnogalacturonan I or homogalacturonan with a high degree of esterification, seemed to be not affected.In conclusion, the early modifications occurring in maize cell walls as a consequence of DCB-habituation involved quantitative and qualitative changes of arabinoxylans, but also other polysaccharides. Thereby some of the changes that took place in the cell walls in order to compensate for the lack of cellulose differed according to the DCB-habituation level, and illustrate the ability of plant cells to adopt appropriate coping strategies depending on the herbicide concentration and length of exposure time.

A novel cell division factor from tobacco 2B-13 cells that induced cell division in auxin-starved tobacco BY-2 cells

Effects of auxin as plant hormones are widespread; in fact in almost all aspects of plant growth and development auxin plays a pivotal role. Although auxin is required for propagating cell division in plant cells, its effect upon cell division is least understood. If auxin is depleted from the culture medium, cultured cells cease to divide. It has been demonstrated in this context that the addition of auxin to auxin-starved nondividing tobacco BY-2 cells induced semisynchronous cell division. On the other hand, there are some cell lines, named habituated cells, that can grow without auxin. The cause and reason for the habituated cells have not been clarified. A habituated cell line named 2B-13 is derived from the tobacco BY-2 cell line, which has been most intensively studied among plant cell lines. When we tried to find the difference between two cell lines of BY-2 and 2B-13 cells, we found that the addition of culture filtrated from the auxin-habituated 2B-13 cells induced semisynchronous cell division in auxin-starved BY-2 cells. The cell division factor (CDF) that is responsible for inducing cell division in auxin-starved BY-2 cells was purified to near-homogeneity by sequential passage through a hydroxyapatite column, a ConA Sepharose column and a Sephadex gel filtration column. The resulting purified fraction appeared as a single band of high molecular weight on sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels by silver staining and was able to induce cell division in auxin-starved BY-2 cells. Identification of the protein by MALD-TOF-MS/MS revealed that it is structurally related to P-glycoprotein from Gossypioides kirkii, which belongs to ATP-binding cassette (ABC)-transporters. The significance of CDF as a possible ABC-transporter is discussed in relationship to auxin-autotrophic growth and auxin-signaling pathway.

Cadmium treatment enhances the production of alkaloid secondary metabolites in Catharanthus roseus

We investigated the effect of cadmium (Cd) on the yield of ajmalicine, a secondary metabolite derived from tryptophan, in the suspension cultures of a fully habituated cell line of Catharanthus roseus. The treatment with Cd concentration ranging from 0.05 to 0.4mM and the duration of 24 to 48h increased ajmalicine production and excretion into culture medium, particularly for cells at the mid-exponential growth phase. For early stationary phase cells and stationary phase cells transferred from the culturing medium to the production medium, we detected less enhancement. Cd treatment in cells at the mid-exponential phase, early stationary phase, and cells transferred from the growth medium to production medium increased the cellular content of tryptophan and tryptamine, the precursors for ajmalicine. The tryptamine level was a major limiting factor for ajmalicine production for cells at the mid-exponential phase. The tryptophan decarboxylase (TDC) converting tryptophan to tryptamine was also affected by Cd. We demonstrated that Cd treatment increased the level of TDC transcript, the cellular tryptamine concentration, and ajmalicine excretion, all contributed to the higher yield of ajmalicine. The increase was more significant in cells with active metabolic status and the cellular tryptamine concentration was the major limiting factor for ajmalicine biosynthesis.

NMR natural abundance estimation of13C-metabolic solutes in normal and habituated sugarbeet cell lines

NMR (nuclear magnetic resonance) spectroscopy was used to identify metabolic solutes in one normal and two habituated sugarbeet cell lines (Beta vulgaris L.altissima) obtained from the same mother strain. This technique was applied to investigate the intracellular naturally occurring13C isotopes (1.1% of total natural carbon) in living sugarbeet suspension cells and perchloric cell extracts. A combination of1H,13C, double-quantum filter correlation spectroscopy, heteronuclear multiple-bond correlation, and heteronuclear multiple-quantum coherence spectra from perchloric cell extracts enabled us to identify the main compounds in the different extract solutions. This was verified by spiking the solutions with small amounts of reference compounds to exclude the influence exerted by pH on the chemical shifts of the different compounds in the1H and13C spectra. The comparison of the three sugarbeet cell lines' NMR spectra showed the presence of sucrose, glucose, and fructose in the three strains. On the other hand, it revealed a strong discrepancy between metabolic solutes. Spectra from the habituated lines showed the presence of glutamine. Some amino acids such as alanine or valine, and unidentified signals corresponding to aromatic rings were only characterized in the habituated nonorganogenic cells. On the basis of these13C NMR data we assumed that the discrepancy between the different sugarbeet cell lines could be due to an increase in the metabolic activity of the habituated cell lines in relation to their autonomous growth.

Differential growth dependency of normal and habituated sugarbeet cell lines upon endogenous ethylene production and exogenous ethylene application

A fully habituated (auxin‐ and cytokinin‐independent) nonorganogenic (HNO) sugarbeet (Beta vulgaris) callus produces very little ethylene as compared with a normal (N) hormone‐requiring callus of the same strain. Both callus types react by growth changes to application of inhibitors of ethylene biosynthesis and ethylene action, of 1‐aminocyclopropane‐1‐carboxylic acid (ACC) as the immediate precursor of ethylene, to transfer from light to darkness, and also to application of exogenous ethylene or an ethylene trapper. This indicates their growth dependency upon their endogenously biosynthesized ethylene and also their sensitivity to exogenous gas. However, the sensitivity was generally higher for the HNO callus producing naturally less ethylene. The weaker reaction of the HNO callus to the exogenous ethylene was attributed to its hyperhydric status (a water layer surrounding the cells). Because low ethylene production appears as a general characteristic of habituated cell lines, the causal and/or consequential relationships of this low ethylene production with other characteristics of habituated tissues (absence of exogenous hormones in the culture media, deficiency of cell differentiation, accumulation of polyamines in neoplastic tissues) are discussed.

Cytokinin oxidase activity in habituated and non-habituated soybean callus

Cytokinin oxidase activity was determined in two cell lines of habituated and two of non-habituated soybean callus. In one habituated cell line cytokinin oxidase activity was higher than in the non-habituated lines, whereas the other habituated line showed lower cytokinin oxidase activity. All four callus lines had the highest cytokinin oxidase activity after being subcultured onto new medium. The activity decreased when the cultures reached the exponential growth phase and became undetectable in the stagnant growth phase. The results suggest that cytokinin oxidase may not play a major role in habituation.

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.

Redox characteristics of normal and habituated cell lines of sugarbeet

ABSTRACTRedox properties of cells of normal hormone‐requiring (N) and habituated (H) sugarbeet calli have been investigated. It was found that H cells at all ages reduced exogenous ferricyanide at a much higher rate than N cells, but they exhibited a lower chemiluminescence either alone or in presence of luminol. The plasma membrane NADH:fer‐ricyanide oxidoreductase measured in vitro was almost identical for the two cell types. This could indicate that the higher reducing power of H cells had an intracellular origin. Among the enzyme activities which could provide electrons, malate dehydrogenase activity was found to be a good candidate, being more active in H cells. The results are discussed in relation to the abnormal structure of the cell wall of habituated cells.

Sterol composition of normal and habituated sugarbeet callus (Beta vulgaris L.Altissima)

A detailed qualitative and quantitative analysis of the sterol content of normal (auxin and cytokinin requiring) and habituated (auxin and cytokinin independent) sugarbeet callus (Beta vulgaris L.altissima) was made using mass spectrometry with gas chromatography. The total sterol content of the two lines did not differ significantly. Δ7-Sterols were the most important class of sterols in the two sugarbeet callus lines, as in allChenopodiaceae. Elevated levels of Δ8-sterols were found in the habituated callus. These sterols are considered to be badly integrated in the membrane of eucaryotic cells. A partial blocking of Δ8-Δ7-isomerase is hypothesized in the habituated cell line.

Protoplast fusion ofCatharanthus roseus cells by electrofusion of chemically-agglutinated protoplasts

Mesophyll derived protoplasts ofCatharanthus roseus cv. Little pinkie were fused with protoplasts derived from an habituated cell line ofC. roseus. Polyethylene glycol was used as agglutinating agent while fusions were induced by square pulses. Best results were obtained by fusing protoplasts from primary leaves with those from three-day-old cell cultures. Adding calcium ions considerably enhanced heterofusion rate. Good cell viabilities indicated that this fusion process was not cytotoxic. The heterofusion frequency was up to 10% or more. Most of the heterokaryons were able to regenerate their cell walls and underwent division.

Protective systems against activated oxygen species compared in normal and fully habituated nonorganogenic sugarbeet calluses

The levels of the water-soluble reductants ascorbic acid and glutathione and the activities of the enzymatic antioxidants superoxide dismutase, catalase, ascorbate peroxidase, monodehydroascorbate and dehydroascorbate reductases and glutathione reductase were determined in a fully habituated nonorganogenic sugarbeet callus line (considered a neoplasm) compared with a normal hormone-dependent callus of the same plant. Ascorbic acid was not recovered from either of the two calluses, irrespective of the technique used. Glutathione was titrated at a slightly higher level in the normal callus. Catalase activity was almost nonexistent in the habituated callus. The other enzymes (superoxide dismutase, glutathione reductase, monodehydroascorbate reductase, dehydroascorbate reductase, and ascorbate peroxidase) were found to have higher activities in the habituated callus. The results are interpreted as a higher protection of the neoplastic habituated cells against oxygen-free radicals and hydroperoxide-dependent oxidations. Such strong scavenging properties of the habituated cell line could explain previous results already reported, namely the stimulation of cell division at the expense of cell differentiation.

Isoperoxidases as markers of somatic embryogenesis in carrot cell suspension cultures

Growth, peroxidase activity and isoperoxidase pattern were studied during the growth cycle of 3 cell suspension lines of carrot (Daucus carota L.), an embryogenic, a non‐embryogenic and a habituated cell line. Isoelectric focusing of extracted proteins on agarose gels revealed the isoperoxidase pattern of the embryogenic line to include, among other differences, an isoperoxidase with a pl of pH 7.0 when grown under conditions stimulating embryogenesis. This isoperoxidase (P7.0: EC 1.11.1.7) was present between days 2 and 6 after subculturing, and this period correlates well with the early stages of somatic embryogenesis. This isoenzyme showed very low activity in the non‐embryogenic and habituated cell suspension lines as well as in the embryogenic cell line in the presence of Daucus carota, 2,4–dichlorophenoxyacetic acid. P7.0 could probably be used as a biochemical marker of somatic embryogenesis.

Peptides of normal and variant cells of tobacco

AbstractPolypeptides solubilized from established normal and variant cell lines of Nicotiana tabacum L cv “Wisconsin 38” have been analyzed by one‐dimensional and two‐dimensional gel electrophoresis. There was little variability observed in the polypeptide profile in an established cell line; polypeptides present in different clonal lines of cells, all derived from an initial established cell culture, were very similar, if not identical. However, a large fraction of the observed polypeptides present in cytokinin‐habituated cell lines (up to 3.8% of the total polypeptides analyzed by two‐dimensional gel electrophoresis) were different from those found in the cytokinin‐requiring cells from which they were selected. The habituated nature of the selected cell lines was demonstrated to be epigenetic; tissue cultures that were reisolated from plantlets regenerated from habituated cell lines did require cytokinin. Further observations demonstrate: (1) that epigenetic events that alter a cellular phenotype change the expression of a relatively large number of polypeptides, (2) that a single epigenetic phenotype may be the result of any one of a number of possible patterns of gene expression, and (3) that epigenetic events are not random events.