Isozymes as biochemical and cytochemical markers in embryogenic callus cultures of maize (Zea mays L.)

Isozyme analyses were carried out on protein extracts of non-embryogenic and embryogenic callus fromZea mays L., using polyacrylamide gel electrophoresis. We examined the isozyme patterns of glutamate dehydrogenase, peroxidase and acid phosphatase for their utility as biochemical markers of maize embryogenic callus cultures. These isozyme systems were also used to examine possible correlations between isozymes and different stages of regeneration. The zymograms of peroxidase and glutamate dehydrogenase differed for non-embryogenic and embryogenic callus. Further, some isozymes were correlated with the morphological appearance of the tissue while others seemed to be involved with the duration of the culture period. Using the same enzyme assays on fresh tissue samples we were able to test the three enzymes as cytochemical markers in embryogenic cultures. Glutamate dehydrogenase proved to be most successful to discriminate embryogenic from non-embryogenic cells.     

Glucose-6-phosphate dehydrogenase in tobacco callus strains differing in their growth and their requirement for auxin and cytokinin

The cytokinin-autonomous strain (A_s) of tobacco callus differs from the original cytokinin-dependent strain (D) and from the cytokinin- and auxin-autonomous strain (A₄) by a significantly lower activity of glucose-6-phosphate dehydrogenase (G-6-PDH). Changes in the total G-6-PDH activity were associated with differences in the number of G-6-PDH isozymes. The A_s strain contained only one isozyme, four isozymes were found in D and A₄ strains.     

Superoxide dismutase and peroxidase are coordinately regulated in differentiated and transformed tissues of Nicotiana tabacum

We used a series of normal and Agrobacterium-transformed, bacteria-free tobacco tissue cultures which differ in their levels of histodifferentiation to test the relationship of superoxide dismutase, peroxidase, and catalase to oncogenic transformation and differentiation. When compared with normal callus, tumor callus contained reduced levels of both superoxide dismutase and peroxidase, and a reduced number of isozymes of both enzymes. Teratomas characterized by limited but abnormal differentiation showed increases in superoxide-dismutase activity and isozymes, but levels of peroxidase activity lower than those found in normal callus despite an increase in the number of peroxidase isozymes. A similar disparity between low peroxidase activity and high isozyme number in the shoot suggests that there are increased levels of peroxidase inhibitors or of molecules which interfere with the spectrophotometric assay for peroxidase in more differentiated tissues. As judged by the number of isozymes of both superoxide dismutase and peroxidase in each tissue, the following conclusions are warranted: first, tobacco copper/zinc superoxide dismutases and peroxidases are encoded in several duplicated loci which are regulated independently. Second, transformation is associated with a decrease in both the specific activity and isozyme number of superoxide dismutase. Third, the partial release from the total inhibition of expression of differentiated function exhibited by teratoma is associated with an increase in both the activity and isozyme number of superoxide dismutase. Finally, the expression of superoxide dismutase and peroxidase isozymes appears to be coordinated during differentiation in a manner that is consistent with their role in an integrated mechanism for the removal of reduced oxygen species.     

Biochemical studies of embryogenic and non-embryogenic callus of Cardiospermum halicacabum L

Some physiological and biochemical changes were measured between embryogenic and non-embryogenic callus obtained from Cardiospermum halicacabum. Combination of auxin with cytokinin was more favourable for high amount of callus formation. 2,4-D played a key role in triggering somatic embryo formation. Embryogenic callus had more total carbohydrate and starch contents, total free amino acids, nucleic acids, phenols and ascorbic acid. Non-embryogenic callus exhibited high chlorophyll content, total soluble sugar, protein, ammonia and enzymes like peroxidase and polyphenol oxidase. Thus, the present study indicated that the process of somatic embryogenesis was characterized by some biochemical and physiological changes induced by plant growth regulators.     

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.     

Erythromycin as a tool to investigate the tetrapyrrole biosynthetic pathways in habituated and normal sugarbeet calli

Erythromycin (ERT) has been shown to reduce the 5-aminolevulinic acid (ALA) synthesizing capacity of a normal (N) chlorophyllous sugarbeet callus, grown under light, in contrast to a habituated achlorophyllous non-organogenic (HNO) callus of the same species. Similar effects were obtained on total hemes and on catalase which is a hemoprotein used as marker. The effect of ERT, which is an inhibitor of plastid differentiation and of chlorophyll synthesis, was reversed in the N callus by a supply of glycine and succinate. The compounds are the precursors of ALA synthesized through 5-aminolevulinic acid synthase (ALAS) which is implied in the Shemin pathway. The involvement of ALAS appeared to be favoured when plastids were undifferentiated (HNO callus) or when plastids were inefficient (N callus under darkness or under light after ERT treatment).     

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.     

The effect of sugars on inverse relation between the growth and chlorophy11 synthesis in tobacco tissue

Cytokinin-dependent and cytokinin-autonomous strains of tobacco callus tissue (Nicotiana tabacum L. cv. ‘Wisconsin 38’) were grown on media containing sucrose, glucose and fructose, respectively. The tissues were kept 14 days in darkness and then transferred for 9 days to continuous light after which time the fresh weight and chlorophyll content were estimated. The highest chlorophyll concentration was recorded at sugar levels which were either suboptimal (sucrose in the case of cytokinin-dependent strain) or supraoptimal (all other sugars for both strains and sucrose for the cytokinin-autonomous strain) for tissue growth. The chlorophyll concentration was increased when the tissue was cultured on media containing glucose or fructose,i.e. sugars whioh did not support the growth as well as sucrose. Chlorophyll synthesis in the cytokinin-autonomous strain is significantly lower than in the cytokinin-dependent strain. This difference was independent of either sugar source or concentration. These results support the observed inverse relationship between tissue growth and plastid development and the limited metabolic activity of plastids in cytokinin-autonomous tissues.     

Isozyme modifications during morphogenesis of callus from barley and wheat

Callus cultures fron non-organogenic, young and one-year old, and morphogenic calli were used to assess the value of isozymes analysis for the prediction of morphogenic capacity by studying esterase, peroxidase and acid phosphatase. Basic isozyme patterns of each enzyme for the callus were retained in all the callus stages and in the callus which has differentiated into shoots. With the development of shoot and/or root some conspicuous isozymes appeared for esterase and acid phosphatase and some disappeared for peroxidase. As the isozyme changes became apparent only after shoot or root initiation these enzymes could not be used as markers to distinguish between morphogenic and non-morphogenic calli.     

Isozymes of the glycolytic enzymes in endosperm from developing castor oil seeds

Ion filtration chromatography on diethylaminoethyl-Sephadex A-25 has been used to separate two isozymes each of triose phosphate isomerase, glyceraldehyde 3-phosphate dehydrogenase, glycerate 3-phosphate kinase, enolase, and phosphoglycerate mutase from homogenates of developing castor oil (Ricinus communis L. cv. Baker 296) seeds. Crude plastid fractions, prepared by differential centrifugation, were enriched in one of the isozymes, whereas the cytosolic fractions were enriched in the other. These data (and data published previously) indicate that plastids from developing castor oil seeds have a complete glycolytic pathway and are capable of conversion of hexose phosphate to pyruvate for fatty acid synthesis. The enzymes of this pathway in the plastids are isozymes of the corresponding enzymes located in the cytosol.     

Morphactin-induced changes in the cytokinin effect on tissue and organ cultures ofNicotiana tabacum

Summary The influence of a morphactin, chlorflurenol-methylester (CFM), on the growth, the morphogenesis and the isoelectric peroxidase pattern was investigated in both callus cultures (two different tissue culture strains) and multiple bud cultures ofNicotiana tabacum var.Wisconsin. CFM (range of concentration between 10^–6g/ml and 10^–4g/ml) was applied singly, or in combination with a cytokinin, benzylaminopurine (BAP), or with an auxin, indoleacetic acid (IAA), or with IAA plus BAP.In general, the callus growth was inhibited under the influence of CFM. In some of the experiments carried out in hormone-free media, growth stimulation was observed. Even minimal inhibition or stimulation of the callus growth was always accompanied by characteristic changes in the peroxidase patterns.The following results show the influence of the morphactin CFM on cytokinin effects (endogenous cytokinin or equally the exogenously applied cytokinin, BAP). (1) In the multiple bud cultures, BAP and CFM (both substances combined with IAA) similarly caused inhibition of root formation and stimulation of bud formation. The bands in the peroxidase patterns, characteristic of cytokinin action, were accentuated also of those bud cultures which had been treated with BAP or with CFM. (2) In the callus cultures, the cytokinin characteristics appeared under CFM influence in the peroxidase patterns of one of the tissue culture strains only when CFM was applied in combination with BAP and not in combinations of CFM with IAA.The observed morphactin-induced increase in the cytokinin effects could occur via changes in the hormone level of the tissue.     

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.     

Solasodine Yield in Callus Cultures of Solanum Iaciniatum Ait. Selected for Growth on Hormone-free Medium

Callus autotrophic for both auxin and cytokinin was selectedfrom a hormone-requiring culture of Solanum laciniatum Ait.The control of solasodine yield, callus growth and adventitiousshoot initiation by some exogenous growth regulators and/orlight in the habituated culture was determined. Solasodine concentrationvaried from 0.3 to 1.4 mg g^–1 d.wt. Solanum laciniatum, callus, habituation, solasodine     

Shemin pathway and peroxidase deficiency in a fully habituated and fully heterotrophic non-organogenic sugarbeet callus: an adaptative strategy or the consequence of modified hormonal balances and sensitivities in these cancerous cells? A review and reassessment

There are many arguments for considering a specific fully habituated (auxin and cytokinin-independent) and fully heterotrophic non-organogenic (HNO) sugarbeet callus cell line as terminating a neoplastic progression, and thus to be made of cancerous cells. The similarities with animal tumour and cancer cells are recalled. All types of habituated tissues examined in the literature share at least three common biochemical characteristics: low apparent peroxidase activity, high content of polyamines (PAs) and low production of ethylene. However, results concerning their auxin and cytokinin levels are not consistent. Peroxidase synthesis in the achlorophyllous HNO callus appears to arise from aminolevulinic acid (ALA) synthesis through the Shemin pathway, commonly used by animals and fungi. This pathway is limited by disturbed nitrogen metabolism that diverts glutamate (directly used for ALA synthesis in green higher plants) from the Kreb's cycle into PA synthesis. There is no argument to suggest that the low ethylene production is caused by a competition with PAs for their common precursor, S-adenosylmethionine. The results we report here indicate modified anabolic and catabolic pathways of auxins and cytokinins but also the possibilities of unusual compounds playing similar roles (dehydrodiconiferyl alcohol glucosides, for instance). A higher turnover of PAs is shown in the HNO callus, which could suggest a role for H2O2 and gamma-aminobutyric acid, products or intermediates in the PA catabolic pathway, as secondary messengers. The habituated cells retain some sensitivity towards exogenous auxins and cytokinins. Their increased sensitivity to PAs and ethylene suggests modified hormonal balances for the control of these actively dividing cells.     

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.     

An estimation of the effects of synthetic auxin and cytokinin and the time of their application on some morphological and physiological characteristics of Medicago x varia T. Martyn

The aim of the experiment was to determine the effects of synthetic auxin and cytokinin and the time of their application on some morphological and physiological characteristics of Medicago x varia T. Martyn grown under controlled conditions. The experiment was to check whether an application of exogenous hormones during vegetative and generative stages of the plant had an effect on above-ground mass development, on nitrate reductase activity and on plastid pigments content. Experiment factor was synthetic auxin and cytokinin and the date of their application. Auxin was applied in the form of a synthetic indole-3-butyric acid, while cytokinin was sprayed as synthetic 6-benzylaminopurine. The control plants were treated with distilled water. Depending on the experimental variant, spraying was applied at the sixth true leaf stage and at the first flower bud stage. The research showed that the response of the alfalfa plants to the application of cytokinin and auxin was not uniform. It seems that the most effective was the application of a mixture of them both but only during the vegetative stage.Additionally, cytokinin caused an increase in plastid pigments content in alfalfa leaves. On the other hand, a mixture of auxin and cytokinin triggered the highest nitrate reductase activity in alfalfa roots and raised the ratio of total chlorophyll content to carotenoids. Synthetic auxin caused the decrease of the levels of most parameters compared to the control.     

Peroxidases as Indicators of Growth and Differentiation in Aspen Callus Cultures

Aspen (Populus tremuloides Michx.) callus tissue grown on a synthetic medium containing either an auxin (2,4-dichloro-phenoxyacetic acid) or cytokinin [6-(3-methyl-2-butenylamino) purine] differed in growth rate, total peroxidase activity, peroxidase isoenzyme expression, and in lignin, cell wall sugars and extractive content. Tissue treated with auxin increased more rapidly in fresh weight, but stopped growing sooner than did the cytokinin-treated tissues. Lignification also proceeded more rapidly, and lignin formed a greater fraction of the cell wall weight in auxin-treated tissue. For both treatments, peroxidase activity and growth rate were positively related (r = 0.96). Polyacrylamide gel electrophoresis showed some quantitative, but few qualitative, isoenzyme differences with hormonal treatment and growth rate.     

高等植物葡萄糖-6- 磷酸脱氢酶与6- 磷酸葡萄糖酸脱氢酶基因的不同进化起源

葡萄糖-6-磷酸脱氢酶与6-磷酸葡萄糖酸脱氢酶是植物戊糖磷酸途径中的两个关键酶。在克隆了水稻质体葡萄糖-6-磷酸脱氢酶基因OsG6PDH2和质体6-磷酸葡萄糖脱氢酶基因Os6PGDH2基础上,分析比较了水稻胞质和质体葡萄糖-6-磷酸脱氢酶基因和6-磷酸葡萄糖酸脱氢酶基因的基因结构、表达特性和进化地位。结合双子叶模式植物拟南芥两种酶基因的分析结果,认为高等植物葡萄糖-6-磷酸脱氢酶基因和6-磷酸葡萄糖酸脱氢酶基因在进化方式上截然不同,葡萄糖-6-磷酸脱氢酶的胞质基因与动物和真菌等真核生物具有共同的祖先;6-磷酸葡萄糖酸脱氢酶的胞质酶和质体酶基因都起源于原核生物的内共生。讨论了植物葡萄糖-6-磷酸脱氢酶与6-磷酸葡萄糖酸脱氢酶基因可能的进化模式,为高等植物及质体的进化起源提供了新的资料。     

Effects of the Introduction of Agrobacterium tumefaciens T-DNA ipt Gene in Nicotiana tabacum L. cv. Petit Havana SR1 Plant Cells

Transformation of tobacco leaf discs with the ‘cytokinin’ipt gene yielded several transgenic callus tissue lines, respectiveto the kind of ipt construction present in the A. tumefacienscointegrates. Those calli containing an active ipt gene wereable to grow hormone-autotrophically and showed an increasedendogenous cytokinin level in comparison with controls. Analysisof endogenous IAA level did not allow any quantitative correlationwith the cytokinin content. However, a minimal level of auxinseems to be necessary to obtain hormone-autotrophic growth.Exogenously supplied NAA significantly reduced the endogenouscytokinin content without modifying growth characteristics. The varying chlorophyll content in the different callus lineselicited the study of the ultrastructure of the plastids. Thecontrols contained small plastids, often filled with starchor accumulated vesicles that did not allow observation of theinternal membrane system. The ‘Pssu-ipt’ line, havinga higher cytokinin content, showed plastids with an internalmembrane system consisting of stroma and grana thylakoids, butthis structure was lost during subculture. Swollen thylakoidsappeared, the amount of starch was reduced and vesicles wereaccumulating. (Received November 15, 1990; Accepted March 4, 1991)