Regulation of cytokinin oxidase activity in tobacco callus expressing the T-DNA ipt gene

There are indications that the cytokinin content in transgenic tissues expressing the cytokinin biosynthetic ipt gene is under metabolic control, which prevents the accumulation of cytokinins to lethal levels. The objective of this study was to investigate the relationships between the content of endogenous cytokinins and the activity of cytokinin oxidase (which is believed to be a copper-containing amine oxidase, EC 1.4.3.6.) in ipt transgenic tobacco callus. In addition, the effect of exogenously applied N-benzyladenine (BA) on this relationship was examined. Endogenous cytokinin concentrations were measured in callus of Nicotiana tabacum L. cv. Petit Havana SRI transformed with the ipt of Agrobacterium tumefaciens under the control of a light-inducible promoter and in non-transformed tissue using LC-tandem mass spectrometry. The activity of cytokinin oxidase was estimated by measuring the conversion of [2,8-³H]N⁶-(Δ²-isopentenyl)adenine to [³H]adenine by enzyme preparations in vitro. The 14-day-old ipt-transformed callus contained a 25-fold higher amount of cytokinins as compared to the non-transformed tissue. Mainly zeatin- and dihydrozeatin-types of cytokinins (free bases, ribosides, nucleotides and O-glucosides) accumulated in the ipt transgenic tissue. The cytokinin pool of both ipt-transformed and non-transformed tissues consisted predominantly of cytokinins that are either resistant to cytokinin oxidase attack (nucleotides and O-glucosides of cytokinins and cytokinins bearing N⁶-saturated side chain) or have a low affinity for the enzyme (zeatin and its riboside). The former represented 71.6 and 74.8% and the latter 27.7 and 24.4% of the pool of endogenous cytokinins in ipt-transformed and non-transformed tissues, respectively. Enzyme preparations from ipt-transformed tissue exhibited 1.5-fold higher cytokinin oxidase activity compared with that observed in control tissues. Application of exogenous BA affected the total levels of cytokinins of the two tissue lines in different ways. The cytokinin content increased by 1.7- and 1.5-fold in ipt-transformed tissues 6 and 12 h after BA application, respectively, while it declined in the non-transformed control by 1.6- to 2.0-fold between 3 and 12 h after BA application. The increase in cytokinin content in the ipt callus is due to an increase of zeatin- and dihydrozeatin-type cytokinins (nucleotides, ribosides and free bases) leading to an enhanced accumulation of O-glucosides after 12 h. Following BA treatment, the cytokinin oxidase activity increased up to 1.8-fold in ipt-transformed and 1.6-fold in non-transformed tissues. The levels of isopentenyl-type cytokinins were near the detection limit; however, the enhancement of cytokinin oxidase activity after BA treatment in both tissue lines was correlated with the content of preferred substrate of the enzyme, N⁶-(Δ²-isopentenyl)adenosine.     

Restoration of shooty morphology of a nontumorous mutant of Nicotiana glauca x N. langsdorffii by cytokinin and the isopentenyltransferase gene

The shooty morphology of a nontumorous amphidiploid mutant of Nicotiana glauca Grah. x N. langsdorffii Weinm. was restored by cytokinins, whether exogenously applied or endogenously produced by transformation of the mutant with a transfer DNA (T-DNA) cytokinin-biosynthesis gene (isopentenyltransferase; ipt). Auxins alone did not confer this effect. Similar transformation was not achieved for the parental species. In the case of transformation with the ipt gene, selection of the transformed tissues was based on its hormone-independent growth in the presence of the antibiotic kanamycin. Transformed tissues exhibited a shooty morphology, indistinguishable from that of wildtype genetic tumors N. glauca x N. langsdorffii. This altered phenotype was caused by the presence and constitutive expression of the ipt gene. The insertion and expression of this gene in transformed tissues was confirmed by using the polymerase chain reaction (PCR) technique as well as conventional molecular hybridization analysis. Expression of the ipt gene led to an elevated level of cytokinin in the transformed mutant tissues. This evidence supports the notion that genetic tumors are caused, at least in part, by elevated levels of cytokinin in interspecific hybrids.     

Light-induced expression of ipt from Agrobacterium tumefaciens results in cytokinin accumulation and osmotic stress symptoms in transgenic tobacco

Cytokinins are plant growth regulators that induce shoot formation, inhibit senescence and root growth. Experiments with hydroponically grown tobacco plants, however, indicated that exogenously applied cytokinin led to the accumulation of proline and osmotin. These responses were also associated with environmental stress reactions, such as salt stress, in many plant species. To test whether increased endogenous cytokinin accumulation led to NaCl stress symptoms, the gene ipt from Agrobacterium tumefaciens, encoding isopentenyl transferase, was transformed into Nicotiana tabacum cv. SR-1 under the control of the light-inducible rbcS-3A promoter from pea. In high light (300 mol PPFD m^-2 s^-1), ipt mRNA was detected and zeatin/zeatin glucoside levels were 10-fold higher than in control plants or when transformants were grown in low light (30 mol PPFD m^-2 s^-1). High light treatment was accompanied by increased levels of proline and osmotin when compared to low light grown transformed and untransformed control plants. Elevated in planta cytokinin levels induced responses also stimulated by salt stress, suggesting either common or overlapping signaling pathways are initiated independently by cytokinin and NaCl, setting in motion gene expression normally elicited by developmental processes such as flowering or environmental stress.Abbreviations IPT isopentenyl, transferase - rbcS-3A gene encoding a small subunit protein (SSU) of Rubisco from Pisum sativum - Rubisco ribulose 1,5-bisphosphate carboxylase/oxygenase     

Fruit-specific expression of the A. tumefaciens isopentenyl transferase gene in tomato: effects on fruit ripening and defense-related gene expression in leaves

This paper describes the analysis of tomato plants transformed with a chimeric gene consisting of the promoter region of a fruit specifically expressed tomato gene linked to the ipt gene coding sequences from the Ti plasmid of Agrobacterium tumefaciens. The pattern of expression of this chimeric gene was found to be consistent with the expression of the endogenous fruit-specific gene and consequently, plants expressing the chimeric gene were phenotypically normal until fruit maturation and ripening. A dramatically altered fruit phenotype, islands of green pericarp tissue remaining on otherwise deep red ripe fruit, was then evident in many of the transformed plants. Cytokinin levels in transformed plant fruit tissues were 10 to 100-fold higher than in control fruit. In the leaves of a fruit-bearing transformant, despite a lack of detectable ipt mRNA accumulation, approximately fourfold higher than control leaf levels of cytokinin were detected. It is suggested that cytokinin produced in fruit is being transported to the leaves since accumulation in leaves of PR-1 and chitinase mRNAs, which encode defense-related proteins known to be induced by cytokinin, occurred only when the transformant was reproductively active. Effects of elevated cytokinin levels on tomato fruit gene expression and cellular differentiation processes are also described.     

Controlled Cytokinin Production in Transgenic Tobacco Using a Copper-Inducible Promoter

The cytokinin group of plant hormones regulates aspects of plant growth and development, including the release of lateral buds from apical dominance and the delay of senescence. In this work the native promoter of a cytokinin synthase gene (ipt) was removed and replaced with a Cu-controllable promoter. Tobacco (Nicotiana tabacum L. cv tabacum) transformed with this Cu-inducible ipt gene (Cu-ipt) was morphologically identical to controls under noninductive conditions in almost all lines produced. However, three lines grew in an altered state, which is indicative of cytokinin overproduction and was confirmed by a full cytokinin analysis of one of these lines. The in vitro treatment of morphologically normal Cu-ipt transformants with Cu²⁺ resulted in delayed leaf senescence and an increase in cytokinin concentration in the one line analyzed. In vivo, inductive conditions resulted in a significant release of lateral buds from apical dominance. The morphological changes seen during these experiments may reflect the spatial aspect of control exerted by this gene expression system, namely expression from the root tissue only. These results confirmed that endogenous cytokinin concentrations in tobacco transformants can be temporally and spatially controlled by the induction of ipt gene expression through the Cu-controllable gene-expression system.     

Dynamics of Endogenous IAA and Cytokinins during the Growth Cycle of Soybean Crown Gall and Untransformed Callus

The dynamics of the endogenous IAA and cytokinin levels duringthe growth cycle of two soybean crown gall lines (green andpale) induced by a nop⁺ Agrobacterium tumefaciens C58 strain,were compared with an untransformed soybean callus line. In both transformed tumor lines maximum cytokinin (essentiallyglucosyl-trans-zeatin) levels were attained in the beginningof the exponential growth phase, followed by a drastic decreasejust before the stationary phase was reached. Quantitativelythe green tumor line showed a 2–3 times higher cytokinincontent compared with the pale line. In the untransformed soybeancallus hardly any significant levels of cytokinin could be detected. Analysis of endogenous IAA levels showed no difference betweenthe two tumor lines and the untransformed callus tissue, allshowing a low and constant level throughout the entire growthcycle. The relevance of the endogenous accumulation of phytohormonesin relation to the hormone autotrophic growth of transformedsoybean tissue is discussed. ³ Senior Research Associate Nationaal Fonds WetenschappelijkOnderzoek (N.F.W.O.). ⁴ Recipient of an Instituut voor Wetenschappelijk Onderzoekin Nijverheid en Landbouw (I.W.O.N.L.) grant.     

Effect of Phytohormone Biosynthesis Genes (ipt and iaaM + iaaH) on the Sexual Reproduction of Transgenic Tobacco Plants

Transformation of Nicotiana silvestris Spegar et Comes plants by the ipt or iaaM + iaaH genes changed the hormonal status of plant reproductive organs. The total content of cytokinins and ABA increased, whereas IAA content in the pistils and anthers of the ipt-plants did not change. Reduced fertility of the ipt plants correlated with an elevated cytokinin and ABA contents of their reproductive organs. Pollen tubes of these plants showed defective growth in pistils in situ, and ovaries manifested a low metabolic activity. The transgenic (iaaM + iaaH)-plants were characterized by an elevated IAA content and reduced ABA content, whereas the total content of cytokinins did not change. The fertility of these plants did not differ from that in the wild type.     

Kinetics of Endogenous Cytokinin, IAA and ABA Levels in Relation to the Growth and Morphology of Tobacco Crown Gall Tissue

The kinetics of endogenous cytokinin, IAA and ABA levels duringthe growth cycle of a wild-type tobacco crown gall (W₃₈-B₆S₃)were compared with that of a shoot-inducing (Shi^-) mutant. Inboth tumor types, high IAA and cytokinin (essentially ribosyl-trans-zeatinand its corresponding glucoside) levels were built up by theend of the linear growth phase and maintained during the greaterpart of the exponential growth period. The stationary phasewas preceded by a very drastic decrease in the endogenous levelof both hormones. Quantitatively, the wild-type tumour showed a higher IAA leveland a reduced cytokinin level compared with the Shi^- mutant.No significantly different endogenous ABA pattern was observed.The reduced cytokinin level might correspond to the ratio oftransformed/untransformed cells in the wild-type tumour whereasthe reduced IAA level in the Shi^- mutant may be correlated withthe deletion of gene 2 in the T-DNA of the pGV 2206 Ti plasmid. The elevated cytokinin/IAA ratio induced shooting mainly ofthe untransformed cells in the Shi^- mutant tissue whereas inthe wild-type, the shoot suppression was compatible with thereduced cytokinin/IAA ratio. ⁴Senior Research Associate Nationaal Fonds WetenschappelijkOnderzoek (N.F.W.O.). ⁵Research Associate N.F.W.O. ⁶Recipient of an Instituut voor Wetenschappelijk Onderzoek inNijverheid en Landbouw grant. (Received February 23, 1984; Accepted June 19, 1984)     

The effect of an elevated cytokinin level using the ipt gene and N 6-benzyladenine on single node and intact potato plant tuberization in vitro

Two models of potato (Solanum tuberosum L.) tuberization in vitro (intact plants and single nodes) were used to study the role of cytokinins in this process. We applied hormone in two different ways. The exogenous addition of 10 mg · L^-1 N ⁶-benzyladenine (BA) into the tuberization medium resulted in advanced tuber formation in intact plants, and microtubers appeared 10–20 days earlier than in the experiments in which no cytokinin was supplied. Transformation with the Agrobacterium tumefaciens ipt gene provided potato clones with endogenously elevated cytokinin levels (3–20 times higher zeatin riboside content in different clones). The onset of tuberization in intact ipt-transformed plants with low transgene expression was advanced in comparison with control material, and exogenously applied BA further promoted the tuberization process. On the contrary, tuberization was strongly inhibited in ipt-transformed nodes, and an external increase of the cytokinin level caused complete inhibition of expiant growth. In untransformed (control) nodes cytokinin application resulted in primary and secondary tuber formation, which depended on the BA concentration in cultivation media.Abbreviations BA N ⁶-benzyladenine - PCR polymerase chain reaction - HPLC high performance liquid chromatography - ELISA enzyme-linked immunosorbent assay - NAA -naphthylacetic acid     

The isopentenyl transferase gene is effective as a selectable marker gene for plant transformation in tobacco (Nicotiana tabacum cv. Petite Havana SRI)

 A selection method for transformed cells which does not inhibit regeneration is important for the establishment and optimization of a transformation protocol. We have assessed the 35S-ipt gene from Agrobacterium tumefaciens as a selectable marker gene. The identification of ipt-expressing cells from nontransformed cells enabled morphological selection without the use of kanamycin and also allowed for the elimination of a high proportion of nonexpressing cells. Ipt selection of tobacco leaf discs (Nicotiana tabacum cv. Petite Havana SRI) resulted in a 2.7-fold higher transformation frequency compared to kanamycin selection. Overexpression of the ipt gene favored plant regeneration from transformed cells, and the transformation frequency of the ipt plus kanamycin selection resulted in a 1.6-fold higher transformation frequency than kanamycin selection alone. These results indicate that this procedure might provide a strategy whereby transgenic plants can be efficiently obtained and some of the problems related to the use of antibiotics diminished. Received: 1 November 1999 / Revision received: 26 June 2000 / Accepted: 18 July 2000     

Tobacco plants transformed with the Agrobacterium T-DNA gene 1 contain high amounts of indole-3-acetamide

The T-DNA genes 1 and 2 of the Ti plasmid of Agrobacterium tumefaciens are involved in the biosynthesis of IAA in transformed plant cells. Previously, it has been shown that gene 2 codes for an amidohydrolase able to convert IAM into IAA. We have isolated Nicotiana tabacum regenerates transformed with either gene 1 or genes 6a and 6b of the T-DNA. The tobacco plants transformed with gene 1 contain 500–1000-times more IAM as compared to plants transformed with genes 6a and 6b, and as compared to untransformed control plants. No drastic differences in endogenous IAA concentrations were observed between the three plant types analyzed.     

Compensation for a Mutated Auxin Biosynthesis Gene of Agrobacterium Ti Plasmid A66 in Nicotiana glutinosa Does Not Result from Increased Auxin Accumulation

Nicotiana glutinosa compensated for a mutated tumor-morphology-shooty (tms) (auxin biosynthesis) locus of Agrobacterlum tumefaciens strain A66 and showed the same virulent tumor response to infection by strain A66 or the wild-type strain A6. Cloned cell lines transformed by strains A6 or A66 were fully hormone independent in culture and grew rapidly as friable, unorganized tissues on hormone-free growth medium. Growth of N. glutinosa tumor cells was inhibited by addition of α-naphthaleneacetic acid to the growth medium, and A6- and A66-transformed cells showed similar dose responses to this auxin. On the other hand, A6-transformed cells contained much higher levels of indole-3-acetic acid (IAA) and 1-aminocyclopropane-1-carboxylic acid (ACC) than A66-transformed cells. Differences in IAA and ACC levels in N. glutinosa tumor lines were consistent with the expected activity of the tms locus and were quantitatively similar to results obtained previously with A6- and A66-transformed cells of Nicotiana tabacum, which does not compensate for mutated tms genes. Thus, compensation for mutated tms genes in N. glutinosa did not result from increased auxin accumulation and did not appear to be related to the capacity of this host for auxin biosynthesis.     

Functional Correlation between Endogenous Phytohormone Levels and Hormone Autotrophy of Transformed and Habituated Soybean Cell Lines

We analysed the time course of the endogenous free IAA and cytokininlevels in hormone requiring and hormone autotrophic (both transformedand untransformed) Glycine max. L. Merr. cv. Mandarin tissuecultures. The auxin habituated line showed an enhanced endogenous IAAlevel, whereas the IAA as well as the cytokinin concentrationsin the cytokinin habituated line differed not significantlyfrom the non-habituated hormone requiring soybean callus. It were only the auxin habituated cells that could be inducedto fully habituated cells, from which a pale and a green typewas isolated. The phytohormone autotrophic growth of the paletype was sustained by enhanced IAA levels, whereas the greentype was characterised by elevated cytokinin concentrations. These results on the phytohormone content of partially and fullyhabituated soybean calli were compared with soybean crown galllines and discussed in view of the positive effect of exogenouslyapplied cytokinins on the endogenous IAA levels. ³Recipient of an Instituut voor Wetenschappelijk Onderzoek inNijverheid en Landbouw (I.W.O.N.L.) grant. ⁴Senior Research Associate Nationaal Fonds voor wetenschappelijkOnderzoek (N.F.W.O.). (Received March 25, 1988; Accepted July 7, 1988)     

Inhibition by Ethylene of Auxin-Promotion of Flower Bud Formation in Tobacco Explants Is Absent in Plants Transformed by Agrobacterium rhizogenes

The in vitro regeneration of flower buds was studied in pedicel explants from tobacco (Nicotiana tabacum L., cv Petit Havana) transformed with Agrobacterium rhizogenes, pRi 1855 (agropine type). At a low concentration (0.1 micromolar) of 1-naphthalene-acetic acid, pedicel strips from phenotypically aberrant plants regenerated two to three times more flower buds than explants from untransformed tobacco. Intermediate bud numbers were observed in transformants with a less extreme phenotype. The results can be explained by an increased sensitivity of the transformed explants to auxin with respect to flower bud regeneration. The effect of transformation on the auxin response is fully accounted for by the absence of a negative interaction of endogenous ethylene with 1-naphthaleneacetic acid, a phenomenon normally encountered in untransformed tissues. Three observations led to this conclusion. Application of 1 micromolar AgNO₃ to untransformed explants increased the number of flower buds to the level observed in transformed tissues but had no effect on transformed pedicel strips; exposure to 10 microliters per liter ethylene strongly reduced the response to auxin at all concentrations in untransformed explants but was almost ineffective in the transformed tissues; and endogenous ethylene synthesis occurred at the same rate in both types of explants.     

The Effect of Mutations in the T-DNA Encoded Auxin Pathway on the Endogenous Phytohormone Content in Cloned Nicotiana tabacum Crown Gall Tissues

We analyzed the endogenous auxin and cytokinin levels of clonedNicotiana tabacum SR 1-lines induced either by the wild-typeAgrobacterium tumefaciens C58 strain or by mutants affectedin the T-DNA-encoded IAA biosynthesis pathway. The wild-typeSR1-C58 line contained up to 20 times more IAA than a nontransformedSRI-callus line. The mutant lines affected in gene 1 (iaaM^–)or gene 2 (iaaH^–) contained intermediate levels of IAA. Analysis of the endogenous levels of indole-3-acetamide (IAM)in the nontransformed SR 1 callus line, the wild-type SR1-C58and the two mutant lines confirmed the T-DNA-induced IAA biosynthesispathway in the transformed tumor cells. Supplementing auxinto the mutant lines resulted in complete suppression of theshoot-forming ability, but no changes in the endogenous IAAlevels. There was no marked difference in the cytokinin level betweenthe nontransformed callus line and the wild type tumor line.The two mutant lines, however, showed a 20- to 30-fold highercytokinin level which was not affected by the addition of NAA.The T-DNA encoded hormone biosynthetic pathways are discussedin relation to pathways of the host plant. (Received July 29, 1986; Accepted February 14, 1987)     

Phenotypically normal transgenic T-cyt tobacco plants as a model for the investigation of plant gene expression in response to phytohormonal stress

The tumour-inducing T-DNA gene 4 (T-cyt gene) of the nopaline Ti plasmid pTiC58 was cloned and introduced into tobacco cells by leaf disc transformation using Agrobacterium plasmid vectors. Tobacco shoots exposed to elevated cytokinin levels were unable to develop roots and lacked apical dominance. Using exogenously applied phytohormone manipulations we were able to regenerate morphologically normal transgenic tobacco plants which differed in endogenous cytokinin levels from normal untransformed plants. Although T-cyt gene mRNA levels, as revealed by dot-blot hybridization data, in these rooting plants were only about half those in primary transformed shoots the total amount of cytokinins was much lower than in crown gall tissue or cytokinin-type transformed shoots as reported by others. Nevertheless the cytokinin content in T-cyt plants was about 3 times greater than in control tobacco plants.Elevated cytokinin levels have been shown to change the expression of several plant genes, including some nuclear genes encoding chloroplast proteins. Our results show that the mRNA levels of chloroplast rbcL gene increase in cytokinin-type transgenic tobacco plants as compared with untransformed plants. Data obtained suggest that T-cyt transgenic plants are a good model for studying plant gene activity in different parts of the plant under endogenous cytokinin stress.