Action de la lumiére sur les peroxydases et sur la teneur en composés phénoliques de tissus de feuilles deCichorium intybus L. cultivés in vitro

Three days” illumination of tissues cultured previously in darkness decreases their peroxidase activity and increases their content in phenolic compounds; conversely, when tissues cultured under continuous illumination are placed in darkness for three days, their peroxidase activity increases and their content in phenolic compounds decreases. There is an inverse relationship between the quantity of phenol compound and peroxidase activity. The isoperoxidase pattern is the same in both illuminated and not illuminated tissues.     

Darkness improves growth and delays necrosis in a nonchlorophyllous habituated sugarbeet callus: Biochemical changes

The transfer of light-cultured green normal (N) and white habituated (HNO) sugarbeet callus to darkness reduced the growth of N callus and improved growth and delayed necrosis in the HNO callus. The decrease of dry matter of N callus under darkness was accompanied by a reduced content of carotenoids and by decreased CO₂ fixation, which was compensated by an increased dependency on externally supplied sucrose. The levels of some organic nitrogen compounds such as glutamate, proline, and free polyamines were not affected by transfer to darkness of N or HNO callus. Darkness decreased ethylene emissions in both callus types. In the HNO callus, the sucrose growth dependency and the CO₂ fixation were unaffected by darkness. Chlorophylls were absent both in light and darkness, whereas some carotenoids were accumulated in the HNO callus only in dark conditions. In another connection, a significant increase of peroxidase activity, which did not occur in the N callus, was induced by darkness in the HNO callus. A decreased content of thio-barbituric acid (TBA)-reactive substances was measured in the HNO callus transferred to darkness, whereas an increase was noticed in the N callus placed in the same conditions. These metabolic changes and the reduction of cellular damage in darkness revealed light-induced stress reactions leading to necrosis and to reduced growth of HNO callus. It appeared that darkness allowed the HNO callus to avoid the photooxidation stress. Therefore, the favorable effect of darkness on HNO growth might be explained by the suppression of photooxidative damage due to the absence of carotenoids. The higher peroxidase activity in the HNO callus maintained in darkness raised the problem of heme synthesis in this heterotrophic callus.     

Polyamine levels in relation to growth and NaCl concentration in normal and habituated sugarbeet callus cultures

Abstract. The concentrations of putrescine, spermidine and spermine, the only polyamines detectable in normal and habituated calli of Beta vulgaris L. ssp. altissima , were much higher in the habituated callus than the normal callus, irrespective of experimental conditions. These results suggest that, in normal (tolerant to NaCl) and habituated (sensitive to NaCl) calli, there exists a competition for the common precursor of ethylene and polyamine biosynthesis viz. S-adenosylmethionine. A disequilibrium favouring the synthesis of putrescine and spermidine in the habituated callus might be linked to structural deterioration of the cell membrane following extended culture or severe osmotic stress (68 mol m⁻¹ NaCl). The maintenance of membrane integrity by the normal callus coincides with ethylene production at the expense of polyamine synthesis. In contrast to the habituated callus, the salinity tolerance of the normal callus is accompanied by the accumulation of proline under hypersaline conditions (274mol m⁻³). The important osmoregulatory role played by quaternary ammonium compounds in the-aerial parts of Chenopodiaceae, especially the sugarbeet, is not observed in the calli, these compounds being found in very low concentrations in saline conditions.     

Comparative study of respiratory metabolism in three types of sugarbeet calli,a normal and two habituated ones (organogenic and non-organogenic)

The respiratory metabolism was studied in three types of sugarbeet (Beta vulgaris L. var. altissima) calli: a normal callus (N) and two fully habituated (auxin- and cytokinin-independent) calli, organogenic (HO) and non-organogenic (HNO). Except for the HO callus at day 14, the oxygen consumption rates of the habituated calli were always higher than that of the normal callus throughout the cycle of culture. The maximum activity of the cyanide-resistant pathway (alternative pathway) was much higher in the two habituated calli than in the normal one. By contrast, important differences were found in HNO and HO calli concerning the activity of the cytochrome pathway. In HNO cells, the high activity of this pathway was correlated with a high ATP level while the inverse situation was observed in HO cells. The physiological significance of these results is discussed.     

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     

Effects of iso-osmotic NaCl and mannitol on growth, proline content, and antioxidant defense in Mammillaria gracilis Pfeiff. in vitro-grown cultures

Effects of iso-osmotic concentrations of NaCl and mannitol were studied in Mammilaria gracilis (Cactaceae) in both calli and tumors grown in vitro. In both tissues, relative growth rates were reduced under osmotic stress, which were accompanied by a decrease in both tissue water and K⁺ content. However, growth was inhibited to a lesser extent after exposure to NaCl, when accumulation of Na⁺ ions was observed. In calli, only salinity increased proline content, whereas with tumors proline accumulated after both osmotic stresses. Osmotic stresses also induced oxidative damage in both cactus tissues, although higher oxidative injury was caused by mannitol in calli and by salt in tumors. Low iso-osmotic concentrations of NaCl (75 mM) and mannitol (150 mM) increased peroxidase, ascorbate peroxidase, and esterase activities, whereas elevated catalase activity was recorded only after mannitol treatment in both tissues. High osmotic stress generally decreased enzymatic activities. However, in calli, esterase activity increased in response to high salinity, whereas ascorbate peroxidase activity was enhanced after high mannitol stress. In conclusion, both in vitro-grown cactus tissues were found to be sensitive to osmotic stress caused by either mannitol or NaCl, but accumulation of Na⁺ ions in response to salt somewhat contributed to osmotic adjustment. However, more prominent oxidative damage induced by NaCl compared to mannitol in tumor could be related to ion toxicity. The mechanisms that mediate responses to salt- and mannitol-induced osmotic stresses differed and were dependent on tissue type.     

Growth Conditions in In Vitro Culture Can Induce Oxidative Stress in <Emphasis Type="Italic">Mammillaria gracilis</Emphasis> Tissues

In vitro propagated plants of Mammillaria gracilis Pfeiff. (Cactaceae) develop calli without any exogenous growth regulators. This habituated tissue spontaneously regenerates morphologically normal as well as hyperhydric shoots. In this study, a possible involvement of activated oxygen metabolism in habituation and hyperhydricity in in vitro propagated plants of Mammillaria gracilis Pfeiff. (Cactaceae) was investigated. Significantly higher malondialdehyde (MDA) and carbonyl contents as well as hydrogen peroxide (H₂O₂) production were observed in habituated callus (HC), hyperhydric regenerated shoots (HS), and tumors (TT) in comparison to normal regenerated shoots (NS). Lipoxygenase (LOX) activity showed a similar trend, with a clear increase in activity in HC and HS. The activities of antioxidative enzymes, namely, peroxidase (POX), ascorbate peroxidase (APX), and catalase (CAT), were also higher in HC, HS, and TT, whereas an increase in superoxide dismutase (SOD) activity was observed in HC and HS. The majority of antioxidative isoenzymes were common to all cactus tissues, although a few tissue-specific bands were noticed. Significant decreases in phenylalanine ammonia lyase (PAL) activity, total phenolic content, and lignification were found in HS, HC, and TT in comparison to NS. Our results showed the appearance of a prominent oxidative stress in HC, HS, and TT as well as a strong induction of the antioxidant system indicating that activated oxygen metabolism could be involved in habituation and hyperhydricity and linked to the loss of tissue organization in M. gracilis. B. Balen and M. Tkalec contributed equally to this work.     

Anionic peroxidase production by Arnebia euchroma callus

Arnebia euchroma callus, obtained from the root cell culture of an Iranian native specimen, has gained a doubling time of 63 H after regular subculturing on Linsmaier-Skoog (LS) medium containing sugar (50 g/L), 2,4-dichlorophenoxyacetic acid (10(-6) M), and kinetin (10(-5) M) under darkness at 25°C. Despite the observed somaclonal variations, peroxidase production by the A. euchroma calli has been stable over 4 years under the aforementioned conditions. Isoelectric focusing experiments revealed that the partially purified A. euchroma peroxidases (AePoxs) are mainly anionic with pI values of about 5.5 and 6.6. AePox reaches its optimal activity at 55°C and pH 7.5. Results of the various kinetic studies suggest that AePox belongs to the type III plant peroxidases with no activity for the oxidation of 3-indoleacetic acid, but seems to play a role in the lignin biosynthesis and H(2) O(2) regulation during the proliferation of the A. euchroma cells on LS medium. Comparing the biochemical properties of AePox with horseradish peroxidase and in view of the ease of solid cell culture, the A. euchroma callus could be considered as a source of plant peroxidase for some biotechnological applications.     

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.     

Redox characteristics of normal and habituated cell lines of sugarbeet

Redox 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.     

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     

Relationships among iron deficit-induced potato callus growth inhibition,Fe distribution,chlorosis, and oxidative stress amplified by reduced antioxidative enzyme activities

Callus cultures were used to investigate and delineate responses of potato to iron (Fe) deficiency conditions over different culture durations. The morphological responses included chlorotic symptoms, reduced fresh weight and area of callus growth on Fe-deficient medium compared to calli grown under Fe sufficient conditions. Biochemically, potato calli under Fe deficit exhibited decreases in chlorophyll and carotenoid contents, reduction in activities of antioxidant enzymes (peroxidase, catalase and ascorbate peroxidase), as well as an increase in ferric chelate reductase (FCR) activity, lipid peroxidation, phenolic production and hydrogen peroxide (H₂O₂) level. Perls staining revealed sparse Fe distribution in Fe-deficient callus cells whereas Fe was widely distributed and intensely stained among numerous actively dividing cells in Fe-sufficient calli. These responses of calli to Fe deficiency were more pronounced with prolonged exposure to such stress leading to severe chlorosis and/or death of cells in chlorosis-susceptible calli but potential chlorosis-tolerant callus cells maintained their greenness and viability. Over a prolonged period in culture, significantly positive correlations were found among callus fresh weight, chlorophyll and carotenoid contents, antioxidant enzyme activities and lipid peroxidation as Fe supplies to the medium was increased. FCR activity was strongly correlated in a negative manner with Fe deficiency, chlorophyll content and peroxidase activity. The responses of calli to Fe supply can serve as reliable indicators for detecting chlorosis tolerance and/or nutrient deficiency stress.     

Habituation in sugarbeet callus: auxin content, auxin protectors, peroxidase pattern and inhibitors

A GC-ECD titration of IAA in normal auxin (2,4-D)-requiring and auxin-independent (habituated) sugarbeet callus revealed an equal amount in both tissues. A comparison of the content and pattern (through starch gel electrophoresis) of soluble, membrane and wall peroxidases indicated that normal tissues contained a higher level of isoperoxidases. Normal tissues also were found to contain higher levels of peroxidase inhibitors and auxin protectors. The hypothesized peroxidase-mediated higher rate of auxin destruction in normal sugarbeet callus is supposed to be counterbalanced by the 2,4-D-controlled auxin protectors.     

Acid phosphatase activity of normal and tumor tissue of tobacco grown in vitro and in vivo

Tissue cultures of Nicotiana labacum consisting of green, albino and habituated (normal origin) and teratoma (tomorous origin) were grown under asceptic conditions for 6 to 8 weeks and their extracts were analyzed for phosphatase activity. Comparative enzyme analyses were also made on crude stem extracts of greenhouse-grown normal and tumor tissues of Nicotiana tabacum (var. Wisconsin) and a hybrid (N. glauca × N. langsdorffii).

All the crude extracts showed acid phosphatase activity with a pH optimum at 5.8 to 6.0. The total protein content and enzyme acivity of teratoma tissue (tumor) was higher than that of green, albino or habituated tissue (normal). Similar increased levels were seen in tumor tissue grown in greenhouse in comparison with greenhouse-grown normal tissues. The crude extracts of each of the tissues did not exhibit any qualitative difference in specificity with the 5 different substrates tested; however, differences in the level of activity was observed.

The effect of 4 different culture media was tested on the growth, protein content and acid phosphatase activity of habituated tobacco in tissue culture. Tissues growing in medium containing high salt concentrations showed higher activities than tissues grown in a basal control medium. From the results, it is suggested that although many factors like auxin and other growth factors can influence growth of habituated tobacco tissue, they need not necessarily affect this specific enzyme activity.

     

Isoenzymes of peroxidase and esterase related to morphogenesis in Mammillaria gracillis Pfeiff. tissue culture

In vitro propagated plants of the cactus Mammillaria gracillis Pfeiff. (Cactaceae) spontaneously produced callus. The habituated callus regenerated normal and hyperhydric shoots without the addition of grown regulators. Tumours were obtained by infecting cactus explants with Agrobacterium tumefaciens; the wild strain B6S3 (tumour TW) or with the rooty mutant GV3101 (tumour TR). Both tumour lines grew vigorously, never expressing any morphogenic potential. In this study, cactus shoots, callus, normal and hyperhydric regenerants and TW and TR tumours were compared with regard to peroxidase (EC 1.11.1.7) and esterase activity, and isoenzyme patterns. Guaiacol peroxidase activity was the lowest in the cactus shoots and in the normal regenerants. Callus, hyperhydric regenerants and tumours had peroxidase activity of 6 to 7 times higher. Esterase activity was measured with 1- and 2-naphthylacetate as broad-spectrum substrates. The highest esterase activity was determined in tumours with both substrates. All tissues, except the TR tumour, had higher esterase activity for 2-compared to 1-naphtylacetate. Peroxidase and esterase isoenzyme patterns were not completely identical among the investigated tissues.     

Peroxidase and catalase changes during in vitro adventitious shoot organogenesis from hypocotyls of Albizia odoratissima L.f. (Benth)

Hypocotyls of Albizia odoratissima cultured on shoot induction medium (MS medium with 7.5 μM BAP and 0.5 μM NAA) showed adventitious shoot organogenesis under light with 16 h photoperiod. Similar cultures under total darkness produced non-morphogenic calli. The changes in the specific peroxidase and catalase activity, total protein content and acidic isoperoxidase pattern were compared between the culture showing shoot organogenesis and culture producing non-morphogenic calli. It was found that in vitro shoot bud differentiation is accompanied by an increase of the specific activities of peroxidase and catalase in culture kept under light. In parallel with the above changes the total protein content reached to the maximum level and also a new isoperoxidase (P10) expressed on the 21st day in cultures kept under light. Conversely, culture producing non-morphogenic calli underwent a reverse change in specific peroxidase activity. This change in antioxidant enzyme activities corresponds to the histological observation of shoot bud differentiation in cultures kept under light.     

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.     

Effects of NaCl and iso-osmotic PEG stress on growth,osmolytes accumulation and antioxidant defense in cultured sugarcane cells

In order to discriminate between the ionic and osmotic components of salt stress, sugarcane (Saccharum officinarum L. cv. Co 86032) calli were cultured on media containing NaCl or polyethylene glycol (PEG) 8000 that exerted the same osmotic pressure (−0.7 MPa). PEG stress exposure for 15 days led to significant growth reduction and loss in water content than salt stressed and control tissues. Osmotic adjustment (OA) was observed in callus tissues grown on salt, but was not evident in callus grown on PEG. Oxidative damage to membranes, estimated in terms of accumulation of thiobarbituric acid reactive substances-TBARS and electrolytic leakage was significantly higher in both the stressed calli than the control however, the extent of damage was more in the PEG stressed calli. The stressed callus tissues showed inhibition of ascorbate peroxidase activity, while catalase activity was increased. These results indicate sensitivity of cells to PEG-mediated stress than salt stress and differences in their OA to these two stress conditions. The sensitivity to the osmotic stress indicate that expression of the stress tolerance response requires the coordinated action of different tissues in a plant and hence was not expressed at the cellular level.     

Identification of a cell wall peroxidase in red calli of Prunus incisa Thunb.

Red calli occur frequently in callus cultures of Prunus incisa Thunb. Calli that become red in color stop growing and turn brown while calli that remain green continue to grow. This study was carried out to compare the accumulation of antioxidant activity in red and green calli. The anthocyanin content, peroxidase isoforms and peroxidase activity were different in red and green calli. Red calli contained higher levels of anthocyanins, cell wall peroxidase activity and lower soluble peroxidase activity than green calli. A basic cell wall peroxidase (pI 10.0) was present only in red calli. Two acidic peroxidases (pI 6.0 and 6.8) had higher accumulation in green calli than in red calli. In the cell wall fraction of red calli, a peroxidase isoform with an apparent molecular mass of 30 kDa was found. MALDI -TOF mass spectrometry and internal amino acid sequence analysis indicate that this protein has a very high similarity with the cell wall peroxidase of Beta vulgarisL .     

Biochemical Basis of Resistance of Tobacco Callus Tissue Cultures to Hydroxyphenylethylamines

It has been reported that hydroxyphenylethylamines, such as tyramine and octopamine, are toxic to tobacco (Nicotiana tabacum L.) callus cultures grown in the presence of auxins, whereas calli grown in the presence of cytokinins and crown gall cultures are resistant to these amines (P. Christou and K.A. Barton [1989] Plant Physiol 89: 564-568). In an attempt to understand the underlying mechanism of this resistance, we compared the fates of tyramine in tyramine-sensitive and tyramine-resistant tobacco tissue cultures (cv Xanthi nc). The very rapid formation of black-colored oxidation products from tyramine in sensitive tissues suggested that the toxicity might be caused by the oxidation of tyramine by phenol oxidases present in the tissues or released into the medium after subculture. This was confirmed through many indirect procedures (effect of exogenously added tyrosinase, induction of polyphenol oxidase [PPO] activity by auxin, etc.). The study of tyramine structure-activity relationships further suggested that the toxicity of tyramine might be due to the formation of indolequinones after oxidation by PPO. Subculture of calli grown on 2,4-dichlorophenoxyacetic acid in a medium containing benzyladenine triggered a slow decrease in PPO activity and dramatic increases in peroxidase and tyramine hydroxycinnamoyl transferase (THT) activities. THT was undetectable in calli grown on 2,4-dichlorophenoxyacetic acid but very active in tyramine-resistant crown gall cultures. Moreover, when [3H]tyramine was fed in vivo to tyramine-resistant tissues, it was rapidly integrated into cell walls in the wound periderm formed at the periphery of the calli. Both the conjugation of tyramine and its integration into cell walls could compete with the formation of toxic quinones and therefore play a part in the resistance. Thus, it seems likely that the control of the toxicity of hydroxyphenylethylamines by cytokinins results primarily from changes in the metabolism and the compartmentation of these amines.