Reversible, cell-heritable changes during the development of tobacco pith tissues

Cytokinin requiring cells of Nicotiana tabacum L. cv "Havana 425" can be induced in culture to become cytokinin autotrophic. This process is known as cytokinin habituation. Earlier we showed that pith parenchyma tissue consists of inducible cells, which habituate at high rates when treated with cytokinin, and noninducible cells, which remain cytokinin requiring under these conditions. The inducible and noninducible phenotypes are determined states that arise during the development of the tobacco plant and are inherited by individual cells. Here we show that pith tissue of plants regenerated from cloned lines of noninducible cells exhibits the inducible phenotype indicating that noninducible cells, or their descendants, can become inducible. This change in competence for habituation appears to have an epigenetic basis; it is reversible, occurs at high rates, and depends on the developmental state of the cells. The habituated state occurs in two forms that can be distinguished by their difference in developmental potential. Habituated cells derived from inducible pith cells give rise to normal plants whose leaf and pith tissues require cytokinin for growth in culture. In contrast, habituated cells obtained by transferring noninducible cells on media with progressively lower cytokinin concentrations give rise to plants whose leaf and pith tissues exhibit a cytokinin-habituated phenotype in culture.     

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.     

Variation in the Competence of Tobacco Pith Cells for Cytokinin-Habituation in Culture

Pith parenchyma tissues of tobacco sometimes lose their exogenous requirement for a cell division factor such as the cytokinin, kinetin. This process, known as cytokinin habituation, appears to involve epigenetic changes since it is a heritable change in cell phenotype which is directed, regularly reversible, and leaves the cell totipotent. In this report, we show that pith cells in culture consist of at least two types of cytokinin-requiring cells. The first type habituates rapidly under inductive conditions. The second type continues to express the cytokinin-requiring phenotype for many cell generations in culture but retains the capacity for habituation. These findings suggest that pith cells differ in their competence to habituate and that different states of competence are inherited by individual cells.     

Cytokinin Habituation in Juvenile and Flowering Tobacco

The possible link between cytokinin and flowering was examine in tobacco. The degree of cytokinin autotrophy and the competence for cytokinin habitution were measured in callus derived from pith tissue of Nicotiana tabacum cvs. H425 an W38.Explants were taken from internodes at all positions up the stem in juvenile and mature plants. To test whether the competence of cells to form flowers was linked with crtokinin habituation, thin cell layer explants from comparable internodes were tested for their ability to form floral buds. Callus derived from the upper parts of plants showed cytokinin autotrophy whether or not the plants were flowering. Flower buds were formed only on thin cell layer explants from the upper part of plants which were already flowering. Cytokinin habituation and competence to flower are therefore not directly linked although cytokinin habituation could be a prerequisite for meristematic activity and for flowering. Pith from internodes in the lower half of mature pants formed callus which was cytokinin-dependent, although these same internodes in juvenile plants were cytokinin-autotrophic. The ability to form cytokinin-autotrophic callus was therefore greatest in the meristematic regions and was lost as the pith cells aged. Competence to habituate after 35 °C treatment was also shown by pith callus from a few internodes in the middle of the plant below those already forming cytokinin-autotropic callus.     

The induction of cytokinin habituation in primary pith explants of tobacco

Pith parenchyma tissue ofNicotiana tabacum L. cv. Havana 425 becomes cytokinin habituated when incubated at 35°C on an auxin-containing medium. Under these conditions, habituated, hyperplastic nodules appear on the tissues. We used these nodules to estimate the incidence of habituation by a statistical method. The rate of habituation varied with the season. Tissue isolated from plants in the spring habituated approx. 7 times faster than did tissue isolated from plants in winter. The fact that the average rate, >4×10^–3 per cell generation, was 100–1,000 times faster than the rate of somatic mutation inNicotiana species and depended on the physiological state of the tissue provides further evidence that habituation involves epigenetic changes rather than rare, random genetic mutations. We also found that kinetin (6-furfurylaminopurine) induced habituation and that the concentration required depended on the duration of cytokinin treatment. For long incubation times, approx. 6×10^–10 M kinetin, which is about 1,000-fold lower than the concentration optimal for growth of cytokinin-requiring pith tissue, was sufficient to induce habituation. These results support the hypothesis that the habituated state is maintained by a positive feedback loop in which cytokinins either induce their own synthesis or inhibit their own degradation.     

Habituation of wild chimpanzees (Pan troglodytes) of the South Group at Taï Forest, Côte d'Ivoire: empirical measure of progress

The last part of the chimpanzee (Pan troglodytes) habituation process of the Ta? South Group was monitored over 2 years (1994-1996), during which all males and most of the females became habituated to observation by humans. The time needed (5-7 years) to habituate the community was similar to that recorded for the Ta? North Group and is comparable to other populations studied in Africa. Variation emerged in habituation rate: males were habituated earlier than females, and among females, sexually cycling individuals were habituated faster than non-cycling females. Such differences may be a function of both the methods used to find the chimpanzees and the sex of the individual. Reproductive status and individuality may also have influenced habituation rates by affecting the number of contacts required to habituate a chimpanzee to neutral humans.     

Transfer ribonucleic Acid modification and its relationship to tumorous and nontumorous plant growth

Detailed analyses of tRNA hydrolysates from four tissue types of Nicotiana tabacum, pith from intact plants, pith growing in culture, habituated tissue in culture, and crown gall tumor tissue in culture, revealed significant qualitative and quantitative differences in the pattern of methylation. Although pith from intact plants and pith growing in culture possessed seven different methylated nucleosides, only two were found in habituated and tumorous tissues in culture. Four of the five compounds accounting for the difference were tentatively identified as methylated guanosines. Evaluation of results in terms of several parameters, including growth rate, the tumorous state, habituation, tissue culture, and potential for differentiation, indicate that the extent of tRNA methylation may be correlated with the potential for differentiation of a particular tissue.     

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     

Early habituation of maize (Zea mays) suspension‐cultured cells to 2,6‐dichlorobenzonitrile is associated with the enhancement of antioxidant status

The cellulose biosynthesis inhibitor 2,6‐dichlorobenzonitrile (DCB) has been widely used to gain insights into cell wall composition and architecture. Studies of changes during early habituation to DCB can provide information on mechanisms that allow tolerance/habituation to DCB. In this context, maize‐cultured cells with a reduced amount of cellulose (～20%) were obtained by stepwise habituation to low DCB concentrations. The results reported here attempt to elucidate the putative role of an antioxidant strategy during incipient habituation. The short‐term exposure to DCB of non‐habituated maize‐cultured cells induced a substantial increase in oxidative damage. Concomitantly, short‐term treated cells presented an increase in class III peroxidase and glutathione S‐transferase activities and total glutathione content. Maize cells habituated to 0.3–1 µM DCB (incipient habituation) were characterized by a reduction in the relative cell growth rate, an enhancement of ascorbate peroxidase and class III peroxidase activities, and a net increment in total glutathione content. Moreover, these cell lines showed increased levels of glutathione S‐transferase activity. Changes in antioxidant/conjugation status enabled 0.3 and 0.5 µM DCB‐habituated cells to control lipid peroxidation levels, but this was not the case of maize cells habituated to 1 μM DCB, which despite showing an increased antioxidant capacity were not capable of reducing the oxidative damage to control levels. The results reported here confirm that exposure and incipient habituation of maize cells to DCB are associated with an enhancement in antioxidant/conjugation activities which could play a role in incipient DCB habituation of maize‐cultured cells.     

Habituation of sexual response in male Heliothis moths

It has been generally hypothesized that habituation mediates the effects of pheromone‐based disruption strategies used in the management of moth pests. The current study demonstrates that pheromone‐mediated sexual response in the tobacco budworm moth, Heliothis virescens (F.) (Lepidoptera: Noctuidae), can in fact be modulated by conditions consistent with the production of habituation. An ethogram was used to measure response strength in a wind tunnel experiment where male moths were allowed to respond freely to one of two different blends of female pheromone in 16 trials over 4 days. Post‐test measures were collected to investigate stimulus specificity and long‐term effects. In conditions appropriate to the formation of habituation, habituation will develop and disrupt male sexual response to female sex pheromone. Males repeatedly exposed to plumes of synthetic pheromone blends display a habituated response lasting up to 96 h. Habituation rate and spontaneous recovery of response strength are greater with less intense stimuli. Additionally, males habituated to one blend express no habituation of sexual response when exposed to a different blend. This indicates a high degree of stimulus specificity, which could facilitate outbreeding, and that moths attend to the configuration of the pheromone blend, not simply to its elements.     

The phenolic profile of maize primary cell wall changes in cellulose-deficient cell cultures

Cultured maize cells habituated to grow in the presence of the cellulose synthesis inhibitor dichlobenil (DCB) have a modified cell wall in which the amounts of cellulose are reduced and the amounts of arabinoxylan increased. This paper examines the contribution of cell wall-esterified hydroxycinnamates to the mechanism of DCB habituation. For this purpose, differences in the phenolic composition of DCB-habituated and non-habituated cell walls, throughout the cell culture cycle and the habituation process were characterized by HPLC. DCB habituation was accompanied by a net enrichment in cell wall phenolics irrespective of the cell culture phase. The amount of monomeric phenolics was 2-fold higher in habituated cell walls. Moreover, habituated cell walls were notably enriched in p-coumaric acid. Dehydrodimers were 5–6-fold enhanced as a result of DCB habituation and the steep increase in 8,5′-diferulic acid in habituated cell walls would suggest that this dehydrodimer plays a role in DCB habituation. In summary, the results obtained indicate that cell wall phenolics increased as a consequence of DCB habituation, and suggest that they would play a role in maintaining the functionality of a cellulose impoverished cell wall.     

Immunocytochemical characterization of the cell walls of bean cell suspensions during habituation and dehabituation to dichlobenil

The effects of the cellulose inhibitor dichlobenil on the cell wall composition and structure during the habituation/dehabituation process of suspension‐cultured bean cells were assessed. A range of techniques were used including cell wall fractionation, sugar analysis, immunofluorescence and fluorochrome labelling of resin‐embedded sections, and immunodot assays (IDAs) of cell wall fractions. The cell walls from bean cell suspensions with initial levels of habituation to dichlobenil had decreased levels of cellulose, but this effect lessened with increasing numbers of subcultures. All cell walls analysed showed calcofluor‐stained appositions. However, in habituated and dehabituated cells, appositions were not recognized by an anticallose antibody. This finding suggested the accumulation of an extracellular polysaccharide different to callose, probably a 1,4‐β‐glucan in these cell lines. Appositions in habituated cells also contained homogalacturonan (HG) with a high degree of methyl esterification (DE), rhamnogalacturonan (RG) and xyloglucan. Habituated cell walls were also enriched in pectins, particularly HG, with a low DE, and RG. The levels of extensin epitope that colocalized with RG in habituated cells also diminished with the increasing number of subcultures. Habituated cells also liberated less extensin into the medium. In habituated cells, a decrease in the cell wall arabinogalactan protein (AGP) labelling was observed both in cell walls and in the culture medium. The increase in the number of subcultures in 0.3 µM dichlobenil was accompanied by an increment in some pectic epitopes (JIM5 and LM5) and a decrease in other pectic and in protein epitopes (JIM7, PAM1, LM6, LM2 and MAC207), indicating a re‐structuring of cell walls throughout the habituation procedure. Dehabituated cells showed an overall composition similar to that of non‐habituated cells, with exception of an increase in glucose in hemicellulosic fractions tightly bound to cellulose. However, these cells also showed reduced levels of extensin and AGP labelling. These differences could be related to the high tolerance to dichlobenil observed in dehabituated cells.     

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 1.5 μM 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.     

CAVITY FORMATION IN WINTER BUDS OF PINUS BANKSIANA

The formation of a cavity between the rib meristem and the pith in winter buds of Pinus banksiana is described. Necrosis of cells at the juncture of the rib meristem and pith begins in November, and by mid-March the necrotic region enlarges, forming a cavity that separates the rib meristem from the pith. The cavity appears to be formed by autolysis, rather than a pulling apart of the cells. The function of the cavity remains speculative.     

Chlorophyllous Nature and Growth Characteristics of Teratoma and Habituated Tissue Cultures of Tobacco

The growth characteristics of three types of tissue cultures of tobacco have been investigated. They are (i) pigmented and differentiated teratoma tissue; (ii) pigmented and non-differentiated habituated tissue and (iii) non-pigmented and non-differentiated habituated tissue. All the tissues were grown on a 10 × White's medium without any exogenous supply of auxin. The comparative growth rate of the tissues was related to their nature of differentiation, viz., either organelle differentiation with chloroplast or organ differentiation with buds, leafy appendages, etc. Teratoma and pigmented habituated tissues had normal chlorophyll and carotenoid contents and showed more favorable growth in light than in the dark. The pigmented tissues showed a higher protein content than the non-pigmented tissues irrespective of whether they were of tumorous or normal origin. The pigmented tissues showed less dependence on sucrose availability in the medium than the non-pigmented tissues. Recovery after sucrose starvation was higher in the pigmented tissues than in the non-pigmented tissues. The results indicate that the growth rate of highly differentiated tissues is much slower than non-differentiated tissues and that the pigmented tissues photosynthesize and survive on reserve food products manufactured as a result of photosynthetic activity.     

Expression of hormone-autotrophy in tissues of plantlets regenerated from habituated callus of Nicotiana bigelovii

Experiments presented in the literature show that habituation, a phenomenon characterized by a newly acquired capacity of in vitro cultured plant cells to produce growth regulatory substances, occurs at high rates, leaves the heritably altered cells totipotent and is regularly reversible. Many evidences show that habituation has an epigenetic basis. We report here that habituation in Nicotiana bigelovii var. quadrivalvis tissue cultures is a persistent character not reversed by plant regeneration under the conditions tested.     

Habituation and dehabituation to dichlobenil: Simply the equivalent of Penélope's weaving and unweaving process?

The habituation of cell cultures to cellulose biosynthesis inhibitors constitutes a valuable method for learning more about the plasticity of plant cell wall composition and structure. The subculture of habituated cells in the absence of an inhibitor (dehabituation) offers complementary information: some habituation-associated modifications revert, whereas others remain, even after longterm (3–5 years) dehabituation processes. However, is dehabituation simply the opposite to the process of habituation, in the same way that the cloth woven by Penélope during the day was unwoven during the night? Principal Component Analysis applied to Fourier Transformed Infrared (FTIR) spectra of cell walls from dichlobenil-habituated and dehabituated bean cell lines has shown that dehabituation follows a different pathway to that of habituation. Principal component loadings show that dehabituated cells have more pectins, but that these display a lower degree of methyl-esterification, than those of habituated ones. Further analysis of cell walls focusing on the first steps of habituation would serve to identify which specific modifications in pectins are responsible to the fine modulation of cell wall architecture observed during the habituation/dehabituation process.Key words: cell-wall, cellulose, dichlobenil, habituation-dehabituation, Fourier transform infrared spectroscopy, principal component analysisThe habituation of cell cultures to the presence of lethal concentrations of cellulose biosynthesis inhibitors illustrates the ability of cells to survive with a modified cell wall and is therefore a valuable experimental technique for gaining an insight into the plasticity of plant cell wall composition and structure. Dichlobenil-habituated cultures usually display some common features: slower growth rates, irregularly shaped cells, a trend to grow in clumps when cultured in suspension and compensation of reduced cell wall cellulose content with other cell wall components.^1–3Most of the cell wall changes induced during the habituation to dichlobenil reverted when cells were dehabituated by culturing them in an inhibitor-free medium.^4–7 However, even in long term (3–5 years) dehabituated cell cultures, some habituation-induced cell wall modifications remain, such as altered extractability of pectins, a decrease in arabinogalactan proteins and hydroxyproline-rich glycoproteins epitopes, and the presence of a soluble β-(1,4)-glucan, although cellulose levels were restored.^5–7 Most remarkably, in addition to these stable changes in cell wall architecture, bean dehabituated cells retained a high capacity to cope with lethal concentrations of dichlobenil, as dehabituated cells were forty times more tolerant to dichlobenil than non-habituated cells.⁵ In an attempt to explain the dichlobenil resistance of dehabituated cells it was found that they had a constitutively increased peroxidase activity, indicating a positive relationship between habituation to dichlobenil and antioxidant capacity.⁷If most of the cell wall modifications induced during the habituation to dichlobenil eventually revert to those of non-habituated cells during the dehabituation process, a question arises: is dehabituation simply the inverse of habituation, in the same way that the cloth woven by Penelope during the day was unwoven during the night, as narrated in Homer''s The Odyssey?Principal Component Analysis applied to Fourier Transformed InfraRed spectra of cell walls has been demonstrated to be a powerful technique for conducting comparative analysis of a wide range of cell wall samples.^3,8 Therefore, a suitable approach to answering this question consists in comparison of cell walls from dichlobenil-habituated and dehabituated bean cell lines using this technique.Clearly, FTIR spectra of cell walls from dehabituated cells with few subcultures in the absence of the herbicide resemble those from cultures habituated to high dichlobenil concentrations.⁵ However, the spectra from cells habituated to low inhibitor concentrations and from cells dehabituated for long periods of time⁷ were more similar to those from non-habituated ones. In fact, when Principal Component Analysis is applied to the entire range, Principal Component 2 (PC2) discriminates between Sh₁₂ (corresponding to cells habituated to high dichlobenil concentration) and the rest of the spectra, which is indicative of the above-mentioned similarity (Fig. 1). Nevertheless, PC1 clearly discriminates between spectra from long-term dehabituated cell walls (located at the positive side) and those from cells habituated to low dichlobenil concentrations (at the negative side). This indicates that progression towards dehabituation follows a different path to that of habituation.Open in a separate windowFigure 1Principal Component Analysis of spectra of cell walls from different calluses. A plot of the first two Principal Components scores is represented based on the FTIR spectra of cell walls from non-habituated cells (Snh, ○), cells habituated to different dichlobenil concentrations (Sh, ▲), and cells previously habituated to 12 µm dichlobenil, with a different number of subcultures in the absence of the herbicide (Sd, ◆). Subindexes indicate dichlobenil concentrations in the growth media of habituated cells (0.3, 0.4 or 12 µm); superindexes indicate number of subcultures in the same media. Arrows indicate the different pathways followed by dichlobenil habituation and dehabituation: black arrows, from non-habituated to habituated cells (habituation), and white arrows, from habituated to non-habituated cells (dehabituation).With the aim of identifying those factors which determine this different pathway, PC1 and PC2 loading factors were analyzed (Fig. 2). This analysis indicated that PC2 (explaining 26.4% of total variance) has a positive correlation with wavenumbers attributed to uronic acids (1,420 and 1,600 cm⁻¹) and galactose (950 cm⁻¹), and a negative correlation with wavenumbers associated with cellulose (1,040, 1,060, 1,175, 1,320 and 1,370 cm⁻¹) and xyloglucan (1,125 cm⁻¹). Thus, Sh₁₂ cell walls (clearly located at the positive side of PC2) are pectin enriched and cellulose/xyloglucan impoverished. As explained above, PC1 discriminates between cell walls from dehabituated cell lines and those from cells habituated to low concentrations of dichlobenil. PC1 (accounting for 42.55% of total variance) has a negative correlation with wavenumbers associated with methylester groups (negative peaks at 1,250 and 1,720 cm⁻¹), and a positive correlation with the so called “fingerprint” region (980–1,200 cm⁻¹). Therefore, cell walls from dehabituated cells (those located at the positive side of PC1) would have lower methyl-esterified pectins when compared with cells habituated to low concentrations of dichlobenil.Open in a separate windowFigure 2Loadings for PC1 and PC2 corresponding to Figure 1. White arrowheads point wavenumbers associated with methyl-esterification; black arrowheads, those associated with cellulose and hemicelluloses, and grey arrowheads indicate wavenumbers associated with uronic acids and galactose.Previous results had revealed that dichlobenil habituated cells experienced a progressive reversion in their cell wall composition when they were subcultured in an inhibitor-free medium, gradually increasing their xyloglucan and cellulose content,^5,6 and that both dichlobenil habituated and dehabituated cells showed changes in the distribution of pectin among cell wall fractions: cell suspensions with a low habituation level had cell walls with a higher amount of pectins, and these were more methyl-esterified.⁶Now, FTIR spectroscopy in association to Principal Component Analysis has shown that, although some of the changes observed in the first steps of habituation and in the last steps of dehabituation are common (i.e., reversion of cellulose content), some other changes affect habituated and dehabituated cells differently, and that these changes involve mainly pectin composition and organization. A more detailed analysis of cell walls focusing on the first steps of habituation will serve to identify which specific modifications are responsible for the differences observed in the pectic component and, consequently, responsible for the fine modulation of cell wall architecture.     

The PhoE porin and transmission of the chemical stimulus for induction of acid resistance (acid habituation) in Escherichia coli

R. J. ROWBURY, M. GOODSON AND A.D. WALLACE. 1992. Escherichia coli K12 becomes resistant to killing by acid (habituates to acid) in a few minutes at pH 5.0. Habituation involves protein synthesis-dependent and -independent stages; both must occur at an habituating pH. The habituation sensor does not detect increased ΔpH (or decreased Δψ) nor an increased difference between pH_o and periplasmic pH but probably detects a fall in either external or periplasmic pH. Phosphate ions inhibit habituation, at any stage, probably by interfering with outer membrane passage of hydrogen ions. Most outer membrane components tested are not required for habituation but phoE deletion mutants habituated poorly and are acid-resistant. Strains derepressed for phoE , in contrast, showed increased acid sensitivity. These and other results suggest that habituation involves hydrogen ions or protonated carriers crossing the outer membrane preferentially via the PhoE pore, a process inhibited by phosphate and other anions. Stimulation by phosphate of the poor growth of E. coli at pH 5.0 is in accord with the above. Acetate did not enhance acid killing of pH 5.0 cells, suggesting that their resistance does not depend on maintaining pH_i near to neutrality at an acidic pH_o level.     

Cytokinesis, cell expansion, and the potential for cytokinin-autonomous growth in tobacco pith

Pith tissue from Nicotiana tabacum L. cv `Maryland Mammoth' or `Wisconsin 38' was isolated, free of vascular tissue, and cultured on a medium containing auxin but no cytokinin. Explants from the apical 1 cm of stem, within the pith rib meristem, initiated callus growth with 100% efficiency. Macroscopically visible callus was evident 5 days after the tissue was isolated, and the cultures grew persistently in the absence of cytokinin. Heat treatment, sometimes used to initiate cytokinin habituation, was not required. Explants from tissue basipetal to the pith rib meristem declined in the frequency of habituation with increasing distance from the shoot apex. Although pith tissue which was growing, in vivo, was more prone than mature tissue to establish cytokinin-habituated callus, the basipetal decline in habituation frequency extended well beyond the zone of cell expansion. Explants from mature pith 40 centimeters or more from the shoot apex grew in the absence of cytokinin with 18% frequency, although the response required at least 2 weeks of culture. Further analysis demonstrated that tissue near the periphery of mature pith was more prone to cytokinin-habituation than tissue from the pith center.     

Responsiveness to sucrose and habituation of the proboscis extension response in honey bees

In honey bees, complex behaviours such as associative learning correlate with responsiveness to sucrose. In these behaviours, the subjective evaluation of a sucrose stimulus influences the behavioural performance. Habituation is a well-known form of non-associative learning. In bees, the proboscis extension response can be habituated by repeatedly stimulating the antennae with a low sucrose concentration. A high sucrose concentration can dishabituate the response. This study tests whether habituation correlates with responsiveness to sucrose in bees of different behavioural states and in bees which are habituated with different sucrose concentrations. Habituation and dishabituation in newly emerged bees, 5-day-old bees and foragers strongly correlated with responsiveness to sucrose. Bees with high responsiveness to sucrose displayed a lower degree of habituation and showed greater dishabituation than bees with low responsiveness. The degree of habituation and dishabituation also depended on the concentration of the habituation stimulus. These experiments demonstrate for the first time in a non-associative learning paradigm that the subjective strength of a sucrose stimulus determines the behavioural performance. Non-associative learning shares this property with associative learning, which suggests that the two processes might rely on similar neural mechanisms.Abbreviations: GRS Gustatory response score - PER Proboscis extension response