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

Summary Fully 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.     

Cold-sensitive expression of cytokinin habituation by tobacco pith cells in culture

Cloned, cytokinin-habituated tissues of Nicotiana tobacum L. cv. Havana 425 are able to grow in culture at 25° C without added cytokinin. These tissues vary in their expression of the habituated phenotype at 16° C. When cytokinin-requiring pith tissues are converted to the habituated state by 35° C treatment, all of the habituated cells are cold sensitive. After several transfers in culture, some of these habituated cells give rise to stable, cold resistant variants. Both phenotypes are inherited by individual cells. Cold sensitive clones at 16° C and non-habituated clones at 16° C as well as 25° C show the same dose response to the cytokinin, kinetin. This suggests that at the physiological level, cold sensitivity results from a decreased production of cell division factors rather than from a decreased affinity of cellular receptors for these factors.Abbreviations CDF Cell division factor(s) - NAA -naphthalencacetic acid     

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

Summary 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.     

Tissue-Specific Variation in the Cytokinin Habituation of Cultured Tobacco Cells

Cells of tobacco pith parenchyma sometimes lose their requirement for an exogenous supply of a cell division factor usually supplied as the synthetic cytokinin, kinetin. This change in phenotype, known as cytokinin habituation, is inherited by individual cells and appears to result from epigenetic changes rather than from rare, random, genetic mutations. We have found that tissues from different regions of the tobacco plant exhibit different states of habituation in culture. Pith tissues, as reported earlier, are usually cytokinin requiring and rapidly shift to the habituated state in culture. Leaf tissues are very slightly habituated and require kinetin for optimal rates of growth. Tissues from the stem-cortex are initially habituated. Both the leaf and cortex phenotypes are inherited by individual cells and persist for many cell generations in culture. These results show that certain tissue-specific phenotypes persist in culture and provide evidence that a process akin to habituation leading to different stable states of cytokinin requirement occurs in normal development.     

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

Summary 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.Abbreviations DQF-COSY double-quantum filter correlation spectroscopy - HO habituated organogenous - HNO habituated nonorganogenous - HMBC heteronuclear multiple-bond correlation - HMQC heteronuclear multiple-quantum coherence - N normal - NMR nuclear magnetic resonance - TSP sodium tetradeutero-3-(trimethylsilyl)-propionate     

Rapid Reversion of Cell-Division-Factor Habituated Cells in Culture

Abstract. The heritable conversion of cell-division factor (CDF)-requiring tobacco pith cells to the CDF-autotrophic state, known as CDF habituation, is a directed process that occurs at very high rates in culture. Here we show that tiny, leafy buds arising from habituated tissues placed on an inductive medium exhibit the non-habituated phenotype. The appearance of these buds was used to estimate the rate at which habituated cells revert. The rate obtained, approx. 4 × 10^-3 per cell generation, is at least 100 to 1,000 fold higher than expected for somatic mutation. Moreover, reversion is a directed process, i.e., it occurs at high rates only under conditions that promote bud formation. These findings provide compelling evidence that habituation of tobacco pith cells involves epigenetic changes rather than rare, random genetic mutations.     

Disturbed sugar metabolism in a fully habituated nonorganogenic callus of Beta vulgaris (L.)

Habituated (H) nonorganogenic sugarbeet callus was found to exhibit a disturbed sugar metabolism. In contrast to cells from normal (N) callus, H cells accumulate glucose and fructose and show an abnormal high fructose/glucose ratio. Moreover, H cells which have decreased wall components, display lower glycolytic enzyme activities (hexose phosphate isomerase and phosphofructokinase) which is compensated by higher activities of the enzymes of the hexose monophosphate pathway (glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase). The disturbed sugar metabolism of the H callus is discussed in relation to a deficiency in H₂O₂ detoxifying systems.Abbreviations 6PG-DH 6-phosphogluconate dehydrogenase - G6P-DH glucose-6-phosphate dehydrogenase - H fully habituated callus - HK hexokinase - HMP hexoses monophosphate - HPI hexose phosphate isomerase - N normal callus - PFK phosphofructokinase     

Habituation to humans affects yolk steroid levels and offspring phenotype in quail

In the field as well as in the laboratory, human-generated stress responses are reduced in adult animals previously habituated to humans in comparison to non-habituated individuals. In birds, yolk steroid levels vary with maternal environment and condition. We tested the hypothesis that the experience of female birds with humans could affect yolk steroids levels and offspring phenotype. Two groups of Japanese quail, one habituated to humans (H) and a second non-habituated (NH), were exposed daily to brief human disturbances. We analysed egg quality, offspring growth, and offspring emotional reactivity. NH females produced eggs with less androgens (testosterone and androstenedione) and more immunoreactive progesterone compared to birds habituated to humans. NH females produced eggs with less yolk, heavier shell and chicks hatching later and being smaller as compared to habituated individuals. A lower emotional reactivity was found in young of NH females compared to young of H females. Thus, human disturbance of the mother triggered different effects on chick phenotype depending on previous experience of mother birds with humans. In addition, we describe for the first time the influence of environmental stimuli on yolk immunoreactive progesterone levels. Our results show that a relatively minor difference in behavioral habituation may have substantial effects on eggs and offspring. This has obvious implications for keeping and handling laboratory animals, for conservation biology and for animal welfare.     

Decreased DNA damage by acid and increased repair of acid-damaged DNA in acid-habituated Escherichia coli

A study of the conjugal transfer of ColV,I-K94 tn10 from acid-treated donors suggested that acid-habituated recipients repair acid-damaged plasmid DNA better than those that are not habituated. The presence of an increased repair activity for acid-damaged DNA in habituated cells was confirmed by isolating pBR322 from acid-treated organisms; habituated cells produced more transformants when transformed by it than did non-habituated ones. Additionally, agarose gel electrophoretic studies of pBR322 DNA isolated from acid-damaged cells and tests of its transforming activity both indicated that plasmid DNA in habituated cells is less damaged by extreme acidity than is that in non-habituated organisms.     

Decreased DNA damage by acid and increased repair of acid-damaged DNA in acid-habituated Escherichia coli

A study of the conjugal transfer of ColV,I-K94 tn 10 from acid-treated donors suggested that acid-habituated recipients repair acid-damaged plasmid DNA better than those that are not habituated. The presence of an increased repair activity for acid-damaged DNA in habituated cells was confirmed by isolating pBR322 from acid-treated organisms; habituated cells produced more transformants when transformed by it than did non-habituated ones. Additionally, agarose gel electrophoretic studies of pBR322 DNA isolated from acid-damaged cells and tests of its transforming activity both indicated that plasmid DNA in habituated cells is less damaged by extreme acidity than is that in non-habituated organisms.     

Studies on the use of toxic precursor analogs of opines to select transformed plant cells

Summary S-(2-aminoethyl-)L-cysteine and L-canavanine were less toxic for octopine-type crown gall tissues that contained lysopine dehydrogenase than for other crown gall or habituated tissues. These analogs are substrates for lysopine dehydrogenase in vitro and in vivo. Thus toxic analogs of amino acid precursors of opines may be useful in selecting for cells that contain an opine dehydrogenase.     

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.     

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

Summary 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.     

The effect of ergosterol, ergocalciferol and cholecalciferol on calcium-controlled peroxidase secretion by sugarbeet cells

Provitamin D₂ (ergosterol), vitamins D₂ and D₃ reduced the calcium-mediated peroxidase secretion in three types of sugarbeet cells in suspension cultures. Vitamin D₂ was the most effective in habituated non-organogenic cells which were the less sensitive to calcium; provitamin D₂ was the most effective in habituated organogenic cells, while normal non-organogenic cells were equally sensitive to the three types of vitamin D. The calcium ionophore A23187 slightly restricted peroxidase release in all cases, except in the habituated organogenic cells in the presence of calcium where it exerted a promotive effect. The inhibiting effect of vitamin D₂ was not counteracted by the ionophore except in this habituated organogenic cell line.     

Hormonal control of 5-bromodeoxyuridine (BUdR) tolerance in crown-gall tumors and habituated tissues

We have found that the unorganized tobacco crown-gall tumor, isolated in our laboratory, and Braun's teratomatic tumor both exhibit the capacity to grow in the presence of high toxic concentrations of BUdR (up to 10^-3 mol·l^-1). Double cytokinin-auxin habituated tissues also exhibit a constitutive BUdR-tolerance. In cytokinin-habituated tissues the BUdR-tolerance seems to depend on the degree of cytokinin autonomy. The presence of exogenous cytokinin significantly increases BUdR-tolerance, and the presence of BUdR suppresses the inhibitory effect of exogenous cytokinin upon habituated tissues. The results clearly show that the disposition of a cell for transient BUdR tolerance requires at least active expression of genes for cytokinin synthesis and that the tolerance becomes permanent when the auxin system is turned on. We conclude that BUdR-tolerance is connected with the establishment of a certain hormonal regulator pattern, depending on the state of cellular differentiation.     

The biosynthesis and wall-binding of hemicelluloses in cellulose-deficient maize cells:An example of metabolic plasticity

Cell-suspension cultures(Zea mays L.,Black Mexican sweet corn) habituated to 2,6-dichlorobenzonitrile(DCB) survive with reduced cellulose owing to hemicellulose network modification. We aimed to de fine the hemicellulose metabolism modifications in DCB-habituated maize cells showing a mild reduction in cellulose at different stages in the culture cycle. Using pulse-chase radiolabeling, we fed habituated and non-habituated cultures with [3H]arabinose,and traced the distribution of 3H-pentose residues between xylans, xyloglucans and other polymers in several cellular compartments for 5 h. Habituated cells were slower taking up exogenous [3H]arabinose. Tritium was incorporated into polysaccharide-bound arabinose and xylose residues, but habituated cells diverted a higher proportion of their new [3H]xylose residues into(hetero) xylans at the expense of xyloglucan synthesis. During logarithmic growth, habituated cells showed slower vesicular traf ficking of polymers,especially xylans. Moreover, habituated cells showed a decrease in the strong wall-binding of all pentose-containing polysaccharides studied; correspondingly, especially in log phase cultures, habituation increased the proportion of 3H-hemicelluloses([3H]xylans and [3H]xyloglucan) sloughed into the medium. These findings could be related to the cel walls' cellulose-deficiency, and consequent reduction in binding sites for hemicelluloses; the data could also re fl ect the habituated cells' reduced capacity to integrate arabinox ylans by extra-protoplasmic phenolic cross-linking, as well as xyloglucans, during wall assembly.     

A comparison between ethylene production, ACC and mACC contents, and hydroperoxide level in normal and habituated sugar beet calli

In contrast to normal hormone, requiring sugar beet callus, habituated auxin- and cytokinin-independent callus of the same plant produces very little ethylene, contains less 1-aminocyclopropane-1-carboxylic acid (ACC) and malonyl-ACC (mACC), has a low capacity to convert ACC into ethylene and has lower levels of hydroperoxides. The low ethylene production is apparently controlled by the rate of ACC synthesis and conversion to mACC, as well as by the activity of the ethylene forming enzyme. The interactions between ethylene and polyamine metabolism are discussed, as well as the possible causal relation between the low level of ethylene and the low degree of differentiation of the habituated cells.     

Auxin-polyamine interaction with the calcium-controlled secretion of peroxidase by sugarbeet cells

Spermidine and ornithine given to normal auxin-requiring cell suspensions of sugarbeet inhibited peroxidase secretion in the absence of Ca²⁺. Habituated (organogenic or not) cells did not respond. Both compounds counteracted the Ca²⁺ - promoted enzyme secretion by three cell lines. Auxins (2,4-D and BSAA) did not modify the extracellular level of peroxidase activity in the absence of Ca²⁺ When Ca²⁺ was added, auxins increased its effect in normal cells and had practically no effect in habituated cells. The inhibitory effect of spermidine and ornithine was somewhat reduced by auxins in normal cells and increased in habituated cells. It was hypothesized that the effect of auxins did not involve the mediation of polyamines and that both types of compounds directly interacted with Ca²⁺ at the membrane level.Abbreviations BSAA [benzo(b)selenienyl-3]acetic acid - 2,4-D 2,4-dichlorophenoxyacetic acid - HNO habituated non-organogenic - HO habituated organogenic - NNO normal non-organogenic     

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.     

Caryological behavior during the first phases of dedifferentiation and habituation inNicotiana bigelovii

Summary Hypocotylar tissues ofNicotiana bigelovii var.quadrivalvis seedlings were induced to dedifferentiate and to habituate after a short treatment with 2,4-D or kinetin.Investigation by cytophotometry and chromosome counts on the nuclear cytology of callus induction and development in minimal medium showed, in addition to diploid cells, an high incidence of aneuploid nuclei. Amitotic phenomena have frequently observed during the culture period. The sequence of nuclear events were not influenced by the hormonal composition of the medium.The origin of these aneuploid cells from nuclear fragmentation processes is hypothesized.The results are discussed in comparison with the cytological status of other habituated and tumorous tissues in plant and animal systems.