Daniel Hagège

Polyamines, Phospholipids, and Peroxides in Normal and Habituated Sugar Beet Calli

Summary Compared to a normal (N) auxin and cytokinin requiring sugar beet callus, the habituated (H) auxin and cytokinin independent callus of the same plant contained the same amount of total phospholipids during the course of culture.The peroxide indices of both calli were quite different with an about 2.5 higher level in the N callus.The H callus contained a higher amount of spermine and spermidine than the N one.The contents of putrescine were quite similar.The H callus was characterized by an about 100% increase of its spermine and spermidine content in the 3–14 days following subculture, just before and during active divisions in this highly meristematic tissue.It is hypothesized that the previously shown low level of ethylene production in the H callus might be due to the high amount of polyamines.These substances might also explain the low level of peroxides in the H callus.

Ethylene Production and Polyamine Content of Fully Habituated Sugarbeet Calli

Summary Compared with a normal (N) auxin- and cytokinin-requiring sugarbeet callus, two habituated (a nonorganogenic HNO and a self-shooting HO) calli, obtained from the same mother strain produced a very low amount of ethylene. Ethylene production by HO callus cells in the presence of 1-aminocyclopropane-1-carboxylic acid (ACC) was increased by about 20 times versus 3-fold for HNO cells. The polyamine content of HNO cells was very high compared with N cells, but that of HO was lower. The results are compared with literature data on hormone autonomous lines and shooty tumors. The possible interactions between ethylene and polyamine metabolism are also discussed.

Habituation of plant cells does not mean insensitivity to plant growth regulators

SummaryFully habituated organogenic and nonorganogenic sugarbeet calluses reacted to application of the synthetic auxin [3-benzo(b) selenienyl] acetic acid by changes in growth and ethylene production. Treatment of fully habituated cells of periwinkle with 2,4-dichlorophenoxyacetic acid led to the decrease of free cytokinin contents (isopentenyl adenine, zeatin riboside, and zeatin) during the late exponential phase of growth. The polyamine contents were also modified and the capacity to biotransform secologanin into ajmalicine was decreased. Treatment of the habituated periwinkle cells with zeatin greatly increased the amount of a polypeptide of 16 kDa; this response was more marked than that displayed by the auxin-dependent line. These data show that hormone-independent calluses and cell suspensions can retain some sensitivity to growth hormones. However, differences of responses were observed between the auxin-dependent lines and the habituated lines.

Chlorophylls and carotenoids in a fully habituated nonorganogenic callus of Beta vulgaris

A fully habituated (H) nonorganogenic sugar beet callus, subcultured in the light, did not contain detectable chlorophyll (Chl) nor carotenoid (Car). It accumulated some Car in the dark. Fluorescence spectra indicated that this H callus also accumulated some protochlorophyllide which, however, was not well integrated into the protochlorophyllide-NADPH-photoreductase complex, and therefore not transformed into chlorophyllide in the light. The H callus showed no variable fluorescence which indicated absence of photosynthesis, and therefore it suggested a full heterotrophic behaviour of this peculiar callus line. A green hormone-dependent callus of the same sugar beet had normal fluorescence spectra and kinetics comparable to those of a green leaf.

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

SummaryThe 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.

Abnormal growth of habituated sugarbeet callus and cell suspensions

SummaryHabituated sugarbeet callus and cell suspension cultures derived therefrom, compared to hormone-dependent normal cultures, exhibit a shorter linear growth phase, although they divide actively. Microscopic observations indicate deficiencies in cell expansion and absence of cell differentiation. Cell expansion apparently is interrupted by a cell “budding” process. Some of the cells seem to be empty due to ballooning out of the protoplasm and the bursting of the cell membrane by defective cell wall development. A low amount of cellulose was confirmed by microspectrophotometric estimation. Such cultures exhibit all the characteristics of vitrified tissue.

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

SummaryHabituation 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.

Ras-related proto-oncogenes are transcripted in leaves and callus from sugarbeet

Summary Northern analysis of RNAs, extracted from normal sugarbeet callus cultures (grown with growth regulators) and from leaves derived from the mother strain, when hybridized with a Xenopus c-Ki- ras probe, reveal the presence of two transcripts in the plant material. The level of RNA detected is slightly higher (approximately 2 fold) in callus culture, when compared with leaves from the mother strain. Involvement of cellular oncogenes in the control of plant cells cultured in vitro is suggested.

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

SummaryA 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.