Purification and characterization of adenine phosphoribosyltransferase from Arabidopsis thaliana

Adenine phosphoribosyltransferase (APRT; EC 2. 4,2. 7) from Arabidopsis thaliana was purified approximately 3800-fold, to apparent homogeneity. The purification procedure involved subjecting a leaf extract to heat denaturation, (NH₄)₂SO₄ precipitation, Sephadex G-25 salt separation, ultracentrifugation and liquid chromatography on Diethylaminoethyl Sephacel, Phenyl Sepharose CL-4B, Blue Sepharose CL-6B and adenosine 5'-monophosphate-Agarose. The purified APRT was a homodimer of approximately 54 kDa and it had a specific activity of approximately 300 μmol (mg total protein)^-1 min^-1. Under standard assay conditions, the temperature optimum for APRT activity was 65°C and the pH optimum was temperature dependent. High enzyme activity was dependent upon the presence of divalent cations (Mn²⁺ or Mg²⁺). In the presence of MnCl₂₊ other divalent cations (Mg²⁺, Ca²⁺, Ba²⁺, Hg²⁺ and Cd²⁺) inhibited the APRT reaction. Kinetic studies indicated that 5-phosphoribose-1-pyrophosphate (PRPP) caused substrate inhibition whereas adenine did not. The K_m for adenine was 4.5±1.5 μ M , the K_m for PRPP was 0.29±0.06 m M and the K_i for PRPP was 1.96±0.45 m M . Assays using radiolabelled cytokinins showed that purified APRT can also catalyze the phosphoribosylation of isopentenyladenine and benzyladenine. The K_m for benzyladenine was approximately 0.73±0.06 m M     

钙离子对细胞分裂素延缓水稻叶片衰老的抑制作用

单独使用细胞分裂素 (BA和 Zeatin,1 0 -9～ 1 0 -5 mol/ L和 Ca2 (1 0 -3 mol/ L)处理水稻离体叶片时 ,二者均对叶片衰老有延缓作用。但当用 Ca2 和细胞分裂素同时处理叶片时 ,细胞分裂素延缓衰老的作用受到 Ca2 的明显抑制。进一步研究表明 ,细胞分裂素和 Ca2 并未协同刺激水稻离体叶片的乙烯生成 ,这样排除了通过乙烯促进叶片衰老的可能性。用可提高细胞质 Ca2 浓度的钙通道载体 A2 31 87处理叶片时 ,可延缓叶片衰老 ;而用可降低胞质 Ca2 浓度的试剂 ,如 EGTA、La Cl3 、Verapamil、氯丙嗪等(1 0 -3 mol/ L)处理叶片时 ,可促进叶片衰老 ,进而排除了细胞分裂素促进 Ca2 的吸收而加快衰老的可能性。     

Immunocytochemical study of cell cycle control by cytokinin in cultured soybean cells

An immunocytochemical method was used to determine the proportion of cells in the DNA synthesis (S phase) of the mitotic cycle in suspension cultures of soybean (Glycine max (L.) Merr. cv. Acme) callus of cotyledonary origin, the stably cytokinin-dependent tissue used in the cytokinin bioassay devised by Carlos O. Miller. A standard cell synchronization protocol involving hydroxyurea was used to demonstrate the applicability of the immunocytochemical method to this cell culture. Cells were brought to mitotic arrest by cytokinin withdrawal, and the cell division cycle was restarted by the addition of cytokinin. We have followed the pattern of resumption of S phase after the readdition of cytokinin. This pattern reveals the existence of three subpopulations of cells in cytokinin-starved cultures, consistent with the occurrence of three cytokinin-requiring events in the cell cycle: one in mitosis, one in S phase, and one in the G₁ phase.Abbreviations BrdU 5-bromo-2-deoxyuridine - DI deionized water - FITC fluorescein isothiocyanate - HU hydroxyurea - l-AOPP l--aminooxy--phenylpropionic acid - LI labeling index - PA polyamine - PI propidium iodide     

Rapid increases in cytokinin concentration in lateral buds of chickpea (Cicer arietinum L.) during release of apical dominance

We examined the role of cytokinins (CKs) in release of apical dominance in lateral buds of chickpea (Cicer arietinum L.). Shoot decapitation or application of CKs (benzyladenine, zeatin or dihydrozeatin) stimulated rapid bud growth. Time-lapse video recording revealed growth initiation within 2 h of application of 200 pmol benzyladenine or within 3 h of decapitation. Endogenous CK content in buds changed little in the first 2 h after shoot decapitation, but significantly increased by 6 h, somewhat later than the initiation of bud growth. The main elevated CK was zeatin riboside, whose content per bud increased 7-fold by 6 h and 25-fold by 24 h. Lesser changes were found in amounts of zeatin and isopentenyl adenine CKs. We have yet to distinguish whether these CKs are imported from the roots via the xylem stream or are synthesised in situ in the buds, but CKs may be part of an endogenous signal involved in lateral bud growth stimulation following shoot decapitation. To our knowledge, this is the first detailed report of CK levels in buds themselves during release of apical dominance. Received: 12 December 1996 / Accepted: 7 January 1997     

Desensitization of fifth instar Spodoptera litura to azadirachtin and neem

The deterrence of azadirachtin, in its pure form and as a constituent of neem seed extract, to fifth instar Spodoptera litura (Fab.) larvae, was measured using cabbage, Brassica oleraceae (L.) var. capitata, leaf disc assays. Paired-choice assays, in which larvae could choose between feeding on a treated (1.3 ng azadirachtin per square cm leaf area) or an untreated leaf disc for 2 h, were conducted at 24 h intervals throughout the fifth instar. In addition, no-choice assays, in which larvae could feed on only one leaf disc (10 ng azadirachtin per square cm leaf area) for 1.5 h, were conducted consecutively over a six hour period at the beginning of the fifth instar. The effects of hunger and habituation on desensitization in our no-choice tests were partitioned. After repeated exposures, larvae became desensitized to pure azadirachtinal in both choice and no-choice tests, but did not desensitize to neem containing the same absolute amount of azadirachtin in choice tests. Hunger was responsible for approximately one third of the desensitization response in the no-choice tests. Sensitivity to azadirachtin was independent of age within the fifth instar.     

Growth regulators differentially affect photosensitivity induced by low temperature and osmotic stresses in germinating white clover seeds

Negative photoblastism induced in white clover seeds at 5°C or by lowered water potential (–0.3 MPa, polyethylene glycol) was affected by ethrel, gibberellin A₃, benzylaminopurine and kinetin treatments. The effects were different for water and temperature stressed seeds. The observed synergistic and additive effects of light and growth regulators confirm the earlier suggestion that there are two different mechanisms involved in the light inhibition of white clover seed germination induced by various adverse environmental conditions.Abbreviations ABA abscisic acid - BAP benzylaminopurine - GA₃ gibberellin A₃ - PEG polyethylene glycol     

Changes of chloroplasts density and heterogeneity during the senescence of barley leaves

The equilibrium density of chloroplasts from barley (Hordeum vulgare L. cv. Hassan) was analyzed by sucrose gradient centrifugation. Natural and detachment-induced leaf senescence were associated with a decrease in density and an increase in heterogeneity of the chloroplast population. Treatments (with growth regulators and light) which retarded or accelerated senescence, respectively, retarded or accelerated chloroplast density decrease. Accelerators as well as retardants of senescence decreased the heterogeneity of the chloroplast population.     

Atypical metabolisms and biochemical cycles imposing the cancerous state on plant cells

The biological, morphological and biochemical characteristics which define plant cancer cells at the end of a neoplasic progression in the absence of pathogens and which distinguish them from tumorous cells are summarized. Such plant cancer cells have in common with animal cancer cells many metabolic disturbances. The present paper reviews the biochemical changes in nitrogen, carbon, sugar and heme metabolisms which contribute to polyamine (PAs) accumulation. It indicates how these changes are interconnected and even form between each other biochemical cycles which likely maintain these cells in their irreversible state. The role of these cycles in the maintenance of such cells under a probable permanent oxidative stress is debated.     

Fully habituated sugarbeet callus: Under permanent stress?

Summary A fully habituated nonorganogenic (HNO) sugarbeet callus line, compared to a normal (hormone dependent) one originated from the same plant exhibits many characteristics of a vitrified tissue and several traits common to animal cancer cells. Four types of biochemical or metabolic characteristics of HNO callus [deficiency of tetrapyrrole-containing compounds; lipid (per)oxidation and malondialdehyde formation; high activity of enzyme protective systems; proline, glutamate, and polyamine accumulation] may be interpreted as responses to stress. The deficiency of tetrapyrrole-containing compounds can be considered an indirect protection against activated forms of oxygen as well as the higher activity of the antioxidant defense mechanisms. This fits with suggestions in the literature that the autonomy associated with plant cancer tissue is explained on the basis of antioxidants as stimulators of cell division and corresponding inhibitors of cell differentiation. Such changes occurring in HNO cells may in turn be responsible for a greater absorption and sensitivity to ammonium ions. An altered nitrogen metabolism leads to proline, glutamate, and polyamine accumulation. Lipid peroxidation and malondialdehyde accumulation rapidly occur in this very sensitive HNO callus in a prolonged culture cycle, which might be related to the appearance of necrosis bands. Thus there are arguments allowing us to consider HNO cells as mutants adapted to some stresses, but resulting changes in their structure might have rendered them still more sensitive to other factors.