2,4-D和激动素对烟草愈伤组织IAA氧化酶和细胞分裂素氧化酶活性的影响

2,4-D和激动素(KT)均显著降低烟草愈伤组织中IAA氧化酶和细胞分裂素氧化酶的活性,KT的影响更显著.在MS中的愈伤组织IAA氧化酶活性最高,MS 2,4-D中的次之,MS KT和MS 2,4-D KT中的最低.愈伤组织在MS中继代6 d时,细胞分裂素氧化酶活性出现明显的高峰,在其它3种培养基中则没有.     

Analysis of Cytokinin Metabolism in ipt Transgenic Tobacco by Liquid Chromatography-Tandem Mass Spectrometry

The endogenous levels of the major, naturally occurring cytokinins in Pisum sativum ribulose-1,5-bisphosphate carboxylase small subunit promoter-isopentenyl transferase gene (Pssu-ipt)-transformed tobacco (Nicotiana tabacum L.) callus were quantified using electrospray-liquid chromatography-tandem mass spectrometry during a 6-week subcultivation period. An ipt gene was expressed under control of a tetracycline-inducible promoter for a more detailed study of cytokinin accumulation and metabolism. Activation of the ipt in both expression systems resulted in the production of mainly zeatin-type cytokinins. No accumulation of isopentenyladenine or isopentenyladenosine was observed. In Pssu-ipt-transformed calli, as well as in the tetracycline-inducible ipt leaves, metabolic inactivation occurred through O-glucoside conjugation. No significant elevation of cytokinin N-glucosides levels was observed. Side-chain reduction to dihydrozeatin-type cytokinins was observed in both systems. The levels of the endogenous cytokinins varied in time and were subject to homeostatic regulatory mechanisms. Feeding experiments of ipt transgenic callus with [3H]isopentenyladenine and [3H]isopentenyladenosine mainly led to labeled adenine-like compounds, which are degradation products from cytokininoxidase activity. Incorporation of radioactivity in zeatin riboside was observed, although to a much lesser extent.     

Root-synthesized cytokinins improve shoot growth and fruit yield in salinized tomato (Solanum lycopersicum L.) plants

Salinity limits crop productivity, in part by decreasing shoot concentrations of the growth-promoting and senescence-delaying hormones cytokinins. Since constitutive cytokinin overproduction may have pleiotropic effects on plant development, two approaches assessed whether specific root-localized transgenic IPT (a key enzyme for cytokinin biosynthesis) gene expression could substantially improve tomato plant growth and yield under salinity: transient root IPT induction (HSP70::IPT) and grafting wild-type (WT) shoots onto a constitutive IPT-expressing rootstock (WT/35S::IPT). Transient root IPT induction increased root, xylem sap, and leaf bioactive cytokinin concentrations 2- to 3-fold without shoot IPT gene expression. Although IPT induction reduced root biomass (by 15%) in control (non-salinized) plants, in salinized plants (100?mM NaCl for 22?d), increased cytokinin concentrations delayed stomatal closure and leaf senescence and almost doubled shoot growth (compared with WT plants), with concomitant increases in the essential nutrient K(+) (20%) and decreases in the toxic ion Na(+) (by 30%) and abscisic acid (by 20-40%) concentrations in transpiring mature leaves. Similarly, WT/35S::IPT plants (scion/rootstock) grown with 75?mM NaCl for 90?d had higher fruit trans-zeatin concentrations (1.5- to 2-fold) and yielded 30% more than WT/non-transformed plants. Enhancing root cytokinin synthesis modified both shoot hormonal and ionic status, thus ameliorating salinity-induced decreases in growth and yield.     

Anther-specific expression of ipt gene in transgenic tobacco and its effect on plant development

Isopentenyl transferase (ipt) gene from Agrobacterium tumefaciens T-DNA was placed under the control of a TA29 promoter which expresses specifically in anther. The chimeric TA29-ipt gene was transferred to tobacco plants. During flowering, mRNA of the ipt gene in the anthers of the transgenic plants accumulated and the level of iPA + iPs increased 3–4-fold in the leaves, petals, pistils, and stamens compared with those in the wild type plants. This cytokinin increase affected various aspects in development indicating that the alterations of endogenous cytokinin level by using anther-specific expression of the TA29-ipt gene affected morphology, floral organ systems and reproductivity of the transgenic plants.     

ipt基因定位表达对转基因烟草育性的影响

杂种优势是生物界的一种普遍现象。作物杂种优势的利用是培育高产、抗逆、优质新品种的重要手段之一。然而 ,常规育种技术获得配套三系的难度很大 ,由此限制了杂种优势利用的发展。近年来应用转基因技术创造雄性不育植株已有不少报道[1- 5] 。有研究表明 ,自然突变的雄性不育株     

Senescence-induced ectopic expression of the A. tumefaciens ipt gene in wheat delays leaf senescence, increases cytokinin content, nitrate influx, and nitrate reductase activity, but does not affect grain yield

The manipulation of cytokinin levels by senescence-regulated expression of the Agrobacterium tumefaciens ipt gene through its control by the Arabidopsis SAG12 (senescence-associated gene 12) promoter is an efficient tool for the prolongation of leaf photosynthetic activity which potentially can affect plant productivity. In the present study, the efficiency of this approach was tested on wheat (Triticum aestivum L.)-a monocarpic plant characterized by a fast switch from vegetative to reproductive growth, and rapid translocation of metabolites from leaves to developing grains after anthesis. When compared with the wild-type (WT) control plants, the SAG12::ipt wheat plants exhibited delayed chlorophyll degradation only when grown under limited nitrogen (N) supply. Ten days after anthesis the content of chlorophyll and bioactive cytokinins of the first (flag) leaf of the transgenic plants was 32% and 65% higher, respectively, than that of the control. There was a progressive increase in nitrate influx and nitrate reductase activity. However, the SAG12::ipt and the WT plants did not show differences in yield-related parameters including number of grains and grain weight. These results suggest that the delay of leaf senescence in wheat also delays the translocation of metabolites from leaves to developing grains, as indicated by higher accumulation of ((15)N-labelled) N in spikes of control compared with transgenic plants prior to anthesis. This delay interferes with the wheat reproductive strategy that is based on a fast programmed translocation of metabolites from the senescing leaves to the reproductive sinks shortly after anthesis.     

Inhibitory effects of elevated endogenous cytokinins on nitrate reductase in ipt-expressing tobacco are eliminated by short-term exposure to benzyladenine

Using a novel system for expressing ipt gene from Agrobacterium tumefaciens in tobacco ( Nicotiana tabacum L., cv. Petit Havana SR1), we were able to grow seedlings and teratoma-like tissue with increased content of cytokinins. This material enabled us to investigate new regulatory aspects of nitrate reduction. We grew control plants and plants with elevated cytokinins on MS media, with or without nitrate and benzyladenine (BA). We determined in vitro nitrate reductase (EC 1.6.6.1) activity (NRA) in this plant material. Initially, we found that ipt -expressing plants always displayed lowered levels of NRA when compared to wild-type SR1 plants. We determined that long-term exposure of tobacco plants and tissue to cytokinins caused up to 60% decrease in NRA. Exposure to 40 m M nitrate was able to induce the activity in such plants 3-fold, increasing the activity in SR1 plants more than 5-fold. We were able to restore wild-type levels of NRA in ipt -expressing plants by simultaneous induction of NR with BA and nitrate. Our results suggest that regulation of NR by nitrate and cytokinin is a result of overlaying cytokinin-driven regulatory processes, with those acting in the short-term having a positive effect on NRA, and those acting over extended periods of time having inhibitory effects on NRA.     

Cytokinin oxidase: Biochemical features and physiological significance

The catabolism of cytokinin in plant tissues appears to be due, in large part, to the activity of a specific enzyme, cytokinin oxidase. This enzyme catalyses the oxidation of cytokinin substrates bearing unsaturated isoprenoid side chains, using molecular oxygen as the oxidant. In general, substrate specificity is highly conserved and cytokinin substrates bearing saturated or cyclic side chains do not serve as substrates for most cytokinin oxidases tested to date. Despite variation in molecular properties of the enzyme from a number of higher plants, oxygen is always required for the reaction. Cytokinin oxidases from several sources have been shown to be glycosylated. Cytokinin oxidase activity appears to be universally inhibited by cytokinin-active urea derivatives. Auxin has been reported to act as an allosteric regulator which increases activity of the enzyme.
Cytokinin oxidase activity is subject to tight regulation. Levels of the enzyme are controlled by a mechanism sensitive to cytokinin supply. The up-regulation of cytokinin oxidase expression in response to exogenous application of cytokinin suggests that the metabolic fate of exogenously applied cytokinins may not accurately mimic that of the endogenous compounds.
Cytokinin oxidase is believed to be a copper-containing amine oxidase (EC 1.4.3.6). Considerable evidence strongly supports a common mechanism for amine oxidases. It is possible that advances in understanding of other amine oxidases could be extrapolated to increase our understanding of cytokinin oxidase at the molecular level. This is discussed with reference to what is currently known about the catalytic mechanism of the enzyme. The possibility of pyrroloquinoline quinone, or a closely related compound, as a redox cofactor of cytokinin oxidase is considered, as are the implications of the glycosylated nature of the enzyme for its regulation and compartmentalisation within the cell.     

Cytokinin oxidase activity and cytokinin content in roots of sunflower under water stress

Contents of trans-zeatin riboside (ZR), dihydrozeatin riboside (DZR) and N6-(delta2-isopentenyl) adenosine (iPA) was quantified by an indirect ELISA using polyclonal antibodies, in the roots, xylem sap and leaves of pot grown sunflower plants subjected to water stress (RWC of leaves approximately 65 per cent). The delivery rates of all three cytokinins decreased significantly under stress. Cytokinin levels also decreased in roots and in leaves of stressed plants. Three-fold increase in cytokinin oxidase activity was observed in stressed roots after polymin P-ammonium sulphate fractionation. Further purification using Con A agarose resulted in elution of protein with cytokinin oxidase activity and was found to be 30 kDa protein on SDS-PAGE.     

Regulation of Cytokinin Oxidase Activity in Callus Tissues of Phaseolus vulgaris L. cv Great Northern

The regulation of cytokinin oxidase activity in callus tissues of Phaseolus vulgaris L. cv Great Northern has been examined using an assay based on the oxidation of N⁶-(Δ²-isopentenyl)adenine-8-¹⁴C (i⁶ Ade-8-¹⁴C) to adenine. Solutions of exogenous cytokinins applied directly to the surface of the callus tissues induced relatively rapid increases in cytokinin oxidase activity. The increase in activity was detectable after 1 hour and continued for about 8 hours, reaching values two- to three-fold higher than the controls. The cytokinin-induced increase in cytokinin oxidase activity was inhibited in tissues pretreated with cordycepin or cycloheximide, suggesting that RNA and protein synthesis may be required for the response. Rifampicin and chloramphenicol, at concentrations that inhibited the growth of Great Northern callus tissues, were ineffective in inhibiting the increase in activity. All cytokinin-active compounds tested, including both substrates and nonsubstrates of cytokinin oxidase, were effective in inducing elevated levels of the enzyme in Great Northern callus tissue. The cytokinin-active urea derivative, Thidiazuron, was as effective as any adenine derivative in inducing this response. The addition of Thidiazuron to the reaction volumes used to assay cytokinin oxidase activity resulted in a marked inhibition of the degradation of the labeled i⁶ Ade-8-¹⁴C substrate. On the basis of this result, it is possible that Thidiazuron may serve as a substrate for cytokinin oxidase, but other mechanisms of inhibition have not yet been excluded.     

Cytokinin oxidase/dehydrogenase genes in barley and wheat: cloning and heterologous expression.

The cloning of two novel genes that encode cytokinin oxidase/dehydrogenase (CKX) in barley is described in this work. Transformation of both genes into Arabidopsis and tobacco showed that at least one of the genes codes for a functional enzyme, as its expression caused a cytokinin-deficient phenotype in the heterologous host plants. Additional cloning of two gene fragments, and an in silico search in the public expressed sequence tag clone databases, revealed the presence of at least 13 more members of the CKX gene family in barley and wheat. The expression of three selected barley genes was analyzed by RT-PCR and found to be organ-specific with peak expression in mature kernels. One barley CKX (HvCKX2) was characterized in detail after heterologous expression in tobacco. Interestingly, this enzyme shows a pH optimum at 4.5 and a preference for cytokinin ribosides as substrates, which may indicate its vacuolar targeting. Different substrate specificities, and the pH profiles of cytokinin-degrading enzymes extracted from different barley tissues, are also presented.     

Effects on Fertility in Transgenic Tobacco by Localized Expression of ipt Gene

The isopenteryl transferase (ipt) gene from Agrobacterium tumefaciens (Smith et Townsend) Conn was driven under the tobacco TA29 promoter and introduced into tobacco ( Nicotiana tabacum L.) plants by A. tumefaciens mediated transformation. PCR and Southern blot analysis confirmed that the ipt gene has integrated into the genomes of tobacco plants. The expression pattern of this chimeric TA29-ipt gene in the transgenic plants was studied, and the endogenous cytokinin level in different organs was assayed by ELISA method. The results showed that the cytokinin content in the androecium of transgenic plants increased 3-4 times as compared with the control, and some changes of the fertility of the TA29-ipt transgenic plants have been observed.     

Cytokinin biosynthesis in cultured rootless tobacco plants

Biosynthesis of cytokinin in shoots was examined by growing rootless tobacco (Nicotiana tabacum) plants in vitro. The rootless plants were originated by culturing tobacco callus on a high cytokinin-low auxin medium to induce the formation of plantlets which were then grown on medium without exogenous cytokinin and auxin. The rootless plants supplied with [(14)C]adenine synthesized ethanol-ethyl acetate-water-soluble radioactive components, portions of which had the same chromatographic and electrophoretic mobilities as N(6)-(Delta(2)-isopentenyl)adenine, N(6)-(Delta(2)-isopentenyl)adenosine, 6-(4-hydroxy-3-methyl-2-butenylamino)purine and 6-(4-hydroxy-3-methyl-2-butenylamino)-9-beta-d-ribofuranosylpurine. The total amount of these four major cytokinins was estimated to be present at a concentration of 14 to 23 nanomoles per kilogram of rootless plant. These data indicate that adenine serves as a precursor of the purine moiety of cytokinin molecules and that the cytokinin biosynthetic sites are also located in the shoot in addition to the presumed root sites.