Development of transgenic tobacco harboring a zeatin O-glucosyltransferase gene from Phaseolus

Summary Zeatin and its derivatives are major consituents of higher plant cytokinins. Metabolic steps modifying the isoprenoid side chain, such as O-glycosylation, are expected to have a direct bearing on cytokinin-mediated processes. To examine this possibility, transgenic tobacco plants were generated harboring a gene (ZOG1) encoding a zeatin O-glucosyltransferase from Phaseolus lunatus under the control of a constitutive (35S) and an inducible (Tet) promoter. The presence of the transgene resulted in elevated enzyme production and conversion of exogenous zeatin to its O-glucoside, confirming the expression of the ZOG1 gene in transgenic plants. Endogenous O-glucosylzeatin was increased from less than 1 pmol per g fresh weight in leaves and roots of controls to 26 and 68 pmol per g fresh weight in leaves and roots of 35S-ZOG1 transformants, respectively. In cytokinin/auxin interaction experiments, Tet-ZOG1 leaf discs, in the presence of tetracycline, required 10-fold higher zeatin concentrations for the formation of shoots and callus than the controls. In 35S-ZOG1 plants, developmental changes included adventitious root formation on the lower stems, shorter stature, and axillary shoot growth. Thus, increased zeatin O-glucosylation in detached, cytokinin-dependent tissues leads to a shift in the response to exogenous zeatin indicative of cytokinin sequestering. In whole plants the effect can simulate a reduction or a rise in cytokinin activity depending on the tissue and stage of development. The use of tissue- and stagespecific promoters in the future will allow more precise analyses and targeted growth alterations.     

Promotion of flowering and morphological alterations in Atropa belladonna transformed with a CaMV 35S-rolC chimeric gene of the Ri plasmid

Summary Kanamycin-resistant plants of belladonna (Atropa belladonna) were obtained after Agrobacterium mediated transformation. When a rolC gene, which is one of the loci located on Ri plasmid of Agrobacterium rhizogenes, was co-introduced with a kanamycin resistant (NPT II) gene under control of a cauliflower mosaic virus 35S promoter, the rolC gene was expressed strongly in leaves, flowers, stems and roots. The transformed plants exhibited dramatic promotion of flowering, reduced apical dominance, pale and lanceolated leaves and smaller flowers. On the other hand, when native rolC gene was co-introduced with NPT II, the transgenic plants obtained did not exhibit the altered phenotypes observed in 35S-rolC transformants, and the expression level of the rolC gene was much lower than in 35S-rolC transformants. These results suggest that the morphological changes in transgenic Atropa belladonna were related to the degree of expression of the rolC gene.Abbreviations native rolC rolC gene under control of its own promoter - 35S-rolC rolC gene under control of a cauliflower mosaic viras 35S promoter     

Endogenous cytokinins as biochemical markers of rubber-tree (Hevea brasiliensis) clone rejuvenation

The endogenous levels of isopentenyladenine, isopentenyladenosine, zeatin and zeatin riboside and the ability forin vitro axillary shoot organogenesis and rhizogenesis were compared between mature and rejuvenated clones ofHevea brasiliensis (Müll. Arg.). Enhancement of thein vitro organogenesis ability of rubber-tree clones following somatic embryogenesis or repeated grafting onto juvenile rootstocks was accompanied by an increase of zeatin riboside levels in shoots used as starting material forin vitro micropropagation. Furthermore, the zeatin level, inin vitro shoots of clones treated byin vitro micrografting, and consequently capable of axillary shoot and root organogenesis, was higher than inin vitro shoots of non treated mature material incapable of in vitro organogenesis. We conclude that the endogenous zeatin-like cytokinin level (free and ribosylated forms) can be considered as a reliable marker for the recovery ofin vitro shoot and root organogenesis after rejuvenating treatments in rubber-tree clones.     

Osmotic pretreatment promotes axillary shooting from cauliflower curd pieces by acting through internal cytokinin level modifications

In vitro propagation of cauliflower has generally been achieved through axillary shoot proliferation of curd explants on Murashige and Skoog (MS) medium supplemented with an auxin and a cytokinin. Recently, it has been shown (Vandemoortele 1999) that a soaking in sucrose (-2 MPa for 24 h) of cauliflower curd explants, before culture without any growth regulator, also induced axillary branching. The later procedure avoids the phenomenon of hyperhydricity in the shoots formed. Axillary shooting obtained by the two methods appears to be mediated by modifications of internal cytokinin levels. The osmotic pretreatment did not influence auxin levels, but induced a zeatin and a zeatin riboside levels increase. Curd explants cultured with the usual procedure (on MS medium supplemented with 5 μmol/L BA and 0.5 μmol/L NAA) showed a zeatin and zeatin riboside levels increase of the same magnitude and a higher one for isopentenyl adenine and isopentenyl adenosine. The modification of the cytokinin status in the curd explants subjected to a short osmotic pretreatment thus should be less favourable for hyperhydricity.     

Introduction of a citrus blight-associated gene into Carrizo citrange [<Emphasis Type="Italic">Citrus sinensis</Emphasis> (L.) Osbc. × <Emphasis Type="Italic">Poncirus trifoliata</Emphasis> (L.) Raf.] by <Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation

The protein p12 accumulates in leaves of trees with citrus blight (CB), a serious decline of unknown cause. The function of p12 is not known, but sequence analysis indicates it may be related to expansins. In studies to determine the function of p12, sense and antisense constructs were used to make transgenic Carrizo citrange using an Agrobacterium-mediated transformation system. Homogeneous -glucuronidase⁺ (GUS⁺) sense and antisense transgenic shoots were regenerated using kanamycin as a selective agent. Twenty-five sense and 45 antisense transgenic shoots were in vivo grafted onto Carrizo citrange for further analyses. In addition, 20 sense and 18 antisense shoots were rooted. The homogeneous GUS⁺ plants contained either the p12 sense or antisense gene (without the intron associated with the gene in untransformed citrus) as shown by PCR and Southern blotting. Northern blots showed the expected RNA in the sense and antisense plants. A protein of identical size and immunoreactivity was observed in seven of nine sense plants but not in nine antisense or non-transgenic plants. At the current stage of growth, there are no visual phenotypic differences between the transgenic and non-transgenic plants. Selected plants will be budded with sweet orange for field evaluation for resistance or susceptibility to CB and general rootstock performance.Abbreviations AS Acetosyringone - CaMV 35S P Cauliflower mosaic virus 35S promoter - CaMV 35S poly A Cauliflower mosaic virus 35S poly A terminator - CB Citrus blight - 2,4-D 2,4-Dichlorophenoxyacetic acid - FMV Figwort mosaic virus - GUS -Glucuronidase - GUS gene uidA - IBA Indole-3-butyric acid - MES 2-(N-Morpholino) ethane sulfonic acid - MSI Inoculation medium - MSP-10M Plasmolysis solution with 10% maltose - MSP-8S Plasmolysis solution with 8% sucrose - NAA -Naphthaleneacetic acid - NOS Nopaline synthase - NP Nopaline synthase promoter - NT Nopaline synthase terminator - NPTII Neomycin phosphotransferase II - p12 Blight-associated protein p12     

Application of Arabidopsis AGAMOUS second intron for the engineered ablation of flower development in transgenic tobacco

To explore a new approach to generating reproductive sterility in transgenic plants, the barnase gene from Bacillus amyloliquefaciens was placed under the control of an 1853-bp nucleotide sequence from the 3′end of the second intron of Arabidopsis AGAMOUS and CaMV 35S (−60) minimal promoter [AG-I-35S (−60)::Barnase], and was introduced into tobacco through transformation mediated by Agrobacterium tumefaciens. All AG-I-35S (−60)::Barnase transgenic plants showed normal vegetative growth and 28% of the transgenic lines displayed complete ablation of flowering. Two transgenic lines, Bar-5 and Bar-15, were 98.1 and 98.4% sterile, respectively, as determined by seed production and germination. When controlled by AG-I-35S (−60) chimeric promoter, barnase mRNA was detected in the reproductive tissues of transgenic tobacco plants, but not in vegetative parts. This study presents the first application of an AG intron sequence in the engineered ablation of sexual reproduction in plants. The AG-I-35S (−60)::Barnase construct can be useful in diminishing pollen and seed formation in plants, providing a novel bisexual sterility strategy for interception of transgene escape and has other potentially commercial use for transgenic engineering.     

Functional characterisation of a cytokinin oxidase gene DSCKX1 in Dendrobium orchid

Cytokinin oxidase plays an important role in the cytokinin regulatory processes. We have cloned a novel putative cytokinin oxidase, DSCKX1 (D endrobium Sonia cytokinin oxidase), by mRNA differential display from shoot apices of Dendrobium Sonia cultured in the presence of BA. The DSCKX1 gene appears to have three alternative splicing forms and its expression of DSCKX1 was induced in a tissue-specific manner by cytokinins. In transgenic orchid plants overexpressing DSCKX1, the elevated level of cytokinin oxidase activity was accompanied by a reduction of cytokinin content. These plants exhibited slow shoot growth with numerous and long roots in vitro. Their calli also showed decreased capability of shoot formation. Conversly, antisense transgenic plants showed rapid proliferation of shoots and inhibition of root growth combined with a higher endogenous cytokinin content than wild-type plants. Thus DSCKX1 appears to play an important role on cytokinin metabolism and the related developmental programmes in orchid.     

Increase of endogenous zeatin riboside by introduction of the ipt gene in wild type and the lateral suppressor mutant of tomato

We studied axillary meristem formation of the lateral suppressor (ls) mutant of tomato after elevating the endogenous cytokinin levels through introduction of the isopentenyltransferase (ipt) gene from Agrobacterium tumefaciens. Growth and development of several transformants were examined during in vitro culture. Transformants exhibited phenotypes varying in severity and were divided into four classes. A number of the ipt transformants had a normal phenotype, as non-transformed plants. Others showed a mild to severe cytokinin-like phenotype. Transformants with a mild phenotype exhibited reduced internode length and reduced root development. Transformants with a severe phenotype showed even shorter internodes, loss of apical dominance, reduction of leaf size, production of callus at the basis of the shoots and absence of root development or development of green non-branching roots. The severity of the phenotype correlated well with the level of ipt gene expression, as measured by northern analysis. Transformants with a severe phenotype also exhibited increased levels of zeatin riboside, but zeatin levels were not elevated. The increase in endogenous zeatin riboside levels in the ls mutant did not restore axillary meristem formation, but sometimes bulbous structures were formed in the initially empty leaf axils. Several adventitious meristems and shoots developed from below the surface of these structures. It is concluded that a reduced level of cytokinins in the ls mutant shoots is not responsible for the absence of axillary meristem formation.     

Expression of the <Emphasis Type="Italic">rol</Emphasis>B gene enhances adventitious root formation in hardwood cuttings of aspen

Summary An elite aspen hybrid (Populus × canescens × P. grandidentata) was transformed with Agrobacterium tumefaciens strain EHA105 that harbored a binary vector (pBI121) carrying the nptII gene under the nos promoter and tandem rolB-uidA (GUS) genes with the CaMV 35S or heat shock promoter. Among 32 independent kanamycin-resistant plants, 25 plants were confirmed by polymerase chain reaction and Southern blot analyses to contain all three genes, whereas five plants contained only nptII or/and uidA genes and two plants had both the rolB and nptII or uidA genes. Integration of the rolB gene significantly increased rooting ability of hardwood cuttings. Heat shock-rolB-transformed plants rooted at significantly higher percentage than the CaMV 35S-rolB-transformed plants. Heat shock treatment further enhanced rooting of heat shock-rolB-transformed plants. Exposure to exogenous auxin did not significantly increase the rooting percentage of transgenic hardwood cuttings, but increased the number of roots induced. This research shows great potential to improve rooting of hardwood cuttings of difficult-to-root woody plants which are commercially important to the horticultural and forestry industry. The transgenic plants with gain-of-function in hardwood-cutting rooting can facilitate research in the understanding of adventitious rooting from hardwood cuttings of recalcitrant woody plants.     

Inheritance of gusA and neo genes in transgenic rice

Inheritance of foreign genes neo and gusA in rice (Oryza sativa L. cv. IR54 and Radon) has been investigated in three different primary (T₀) transformants and their progeny plants. T₀ plants were obtained by co-transforming protoplasts from two different rice suspension cultures with the neomycin phosphotransferase II gene [neo or aph (3) II] and the -glucuronidase gene (uidA or gusA) residing on separate chimeric plasmid constructs. The suspension cultures were derived from callus of immature embryos of indica variety IR54 and japonica variety Radon. One transgenic line of Radon (AR2) contained neo driven by the CaMV 35S promoter and gusA driven by the rice actin promoter. A second Radon line (R3) contained neo driven by the CaMV 35S promoter and gusA driven by a promoter of the rice tungro bacilliform virus. The third transgenic line, IR54-1, contained neo driven by the CaMV 35S promoter and gusA driven by the CaMV 35S.Inheritance of the transgenes in progeny of the transgenic rice was investigated by Southern blot analysis and enzyme assays. Southern blot analysis of genomic DNA showed that, regardless of copy numbers of the transgenes in the plant genome and the fact that the two transgenes resided on two different plasmids before transformation, the introduced gusA and neo genes were stably transmitted from one generation to another and co-inherited together in transgenic rice progeny plants derived from self-pollination. Analysis of GUS and NPT II activities in T₁ to T₂ plants provided evidence that inheritance of the gusA and neo genes was in a Mendelian fashion in one plant line (AR2), and in an irregular fashion in the two other plant lines (R3 and IR54-1). Homozygous progeny plants expressing the gusA and neo genes were obtained in the T₂ generation of AR2, but the homozygous state was not found in the other two lines of transgenic rice.     

The cauliflower mosaic virus (CaMV) 35S promoter sequence alters the level and patterns of activity of adjacent tissue- and organ-specific gene promoters

Here we report the effect of the 35S promoter sequence on activities of the tissue- and organ-specific gene promoters in tobacco plants. In the absence of the 35S promoter sequence the AAP2 promoter is active only in vascular tissues as indicated by expression of the AAP2:GUS gene. With the 35S promoter sequence in the same T-plasmid, transgenic plants exhibit twofold to fivefold increase in AAP2 promoter activity and the promoter becomes active in all tissue types. Transgenic plants hosting the ovary-specific AGL5:iaaM gene (iaaM coding an auxin biosynthetic gene) showed a wild-type phenotype except production of seedless fruits, whereas plants hosting the AGL5:iaaM gene along with the 35S promoter sequence showed drastic morphological alterations. RT-PCR analysis confirms that the phenotype was caused by activation of the AGL5:iaaM gene in non-ovary organs including roots, stems and flowers. When the pollen-, ovule- and early embryo-specific PAB5:barnase gene (barnase coding a RNase gene) was transformed, the presence of 35S promoter sequence drastically reduced transformation efficiencies. However, the transformation efficiencies were restored in the absence of 35S promoter, indicating that the 35S promoter might activate the expression of PAB5:barnase in non-reproductive organs such as calli and shoot primordia. Furthermore, if the 35S promoter sequence was replaced with the NOS promoter sequence, no alteration in AAP2, AGL5 or PAB5 promoter activities was observed. Our results demonstrate that the 35S promoter sequence can convert an adjacent tissue- and organ-specific gene promoter into a globally active promoter. Xuelian Zheng and Wei Deng contributed equally to this work and are considered co-first authors.     

Two Agrobacterium tumefaciens genes for cytokinin biosynthesis: Ti plasmid-coded isopentenyltransferases adapted for function in prokaryotic or eukaryotic cells

Summary Tzs and ipt are two Ti plasmid genes coding for proteins with isopentenyltransferase (IPT) activity in vitro. We cloned both genes for protein expression in Escherichia coli and in Agrobacterium tumefaciens, and we investigated differences between the two genes by analysing the properties of the proteins in vitro and in vivo. In vitro, extracts with tzs or ipt-coded proteins had high IPT activity, and the enzymes were identical in most properties. The most important difference was detected in vivo: the tzs-encoded protein was very active in cytokinin production, while the ipt protein required overexpression in order to obtain measurable activity in bacteria. In both cases, rans-zeatin was the major product of the gene activity. Formation of this cytokinin requires a hydroxylase function in addition to the IPT reaction. No such activity could be ascribed to tzs or ipt-encoded proteins in vitro or in vivo, but cytokinin hydroxylase activity was detected in cells and extracts of E. coli, regardless of the presence or absence of the cytokinin genes. Based on these results it is proposed that both genes code for a single enzyme activity (isopentenyltransferase), that the genes and proteins are adapted for function either in bacteria (tzs) or in transformed plant cells (ipt), and that in both prokaryotic and eukaryotic cells hydroxylation to trans-zeatin is a function contributed by host enzymes.Abbreviations DMAPP dimethylallylpyrophosphate - iP isopentenyladenine - iPA isopentenyladenosine - iPMP isopentenyladenosine 5-monophosphate - IPT isopentenyltransferase - trans-Z trans-zeatin     

35S-β-glucuronidase gene blocks biological effects of cotransferred iaa genes

The iaaM and iaaH genes of Agrobacterium tumefaciens and Agrobacterium rhizogenes play an important role in crown gall and hairy root disease. The iaaM gene codes for tryptophan monooxygenase which converts tryptophan into indole-3-acetamide (IAM). IAM is converted into the auxin indole-3-acetic acid (IAA) by indoleacetamide hydrolase, encoded by the iaaH gene. In functional studies on the activity of the iaa genes of the TB region of the A. tumefaciens biotype III strain Tm4, the frequently used 35S--glucuronidase (35S-UidA or GUS) marker gene was found to inhibit IAA synthesis and root induction encoded by the TB iaa genes. To exert this inhibition, the 35S-UidA gene must be cotransferred with the iaaH gene. The 35S promoter alone is sufficient to cause the inhibitory effect.     

Agrobacterium‐produced and exogenous cytokinin‐modulated Agrobacterium‐mediated plant transformation

Agrobacterium tumefaciens is a plant pathogenic bacterium that causes neoplastic growths, called ‘crown gall’, via the transfer and integration of transferred DNA (T‐DNA) from the bacterium into the plant genome. We characterized an acetosyringone (AS)‐induced tumour‐inducing (Ti) plasmid gene, tzs (trans‐zeatin synthesizing), that is responsible for the synthesis of the plant hormone cytokinin in nopaline‐type A. tumefaciens strains. The loss of Tzs protein expression and trans‐zeatin secretions by the tzs frameshift (tzs‐fs) mutant is associated with reduced tumorigenesis efficiency on white radish stems and reduced transformation efficiencies on Arabidopsis roots. Complementation of the tzs‐fs mutant with a wild‐type tzs gene restored wild‐type levels of trans‐zeatin secretions and transformation efficiencies. Exogenous application of cytokinin during infection increased the transient transformation efficiency of Arabidopsis roots infected by strains lacking Tzs, which suggests that the lower transformation efficiency resulted from the lack of Agrobacterium‐produced cytokinin. Interestingly, although the tzs‐fs mutant displayed reduced tumorigenesis efficiency on several tested plants, the loss of Tzs enhanced tumorigenesis efficiencies on green pepper and cowpea. These data strongly suggest that Tzs, by synthesizing trans‐zeatin at early stage(s) of the infection process, modulates plant transformation efficiency by A. tumefaciens.     

Development of flooding-tolerant Arabidopsis thaliana by autoregulated cytokinin production

Flooding is one of the most serious environmental stresses that affect plant growth and productivity. Flooding causes premature senescence which results in leaf chlorosis, necrosis, defoliation, cessation of growth and reduced yield. This study was conducted to determine the effects of autoregulated cytokinin production on the flooding tolerance of Arabidopsis thaliana plants. A chimeric gene containing the senescence-specific SAG12 promoter and the ipt gene coding for isopentenyl transferase, a rate-limiting enzyme in the cytokinin biosynthesis pathway, was constructed. The chimeric gene was introduced into Arabidopsis plants by Agrobacterium-mediated vacuum infiltration. Four transgenic lines were chosen for flooding tolerance determinations. DNA hybridization analysis and PCR confirmed that all four of the transgenic lines carried the ipt gene. The segregation of kanamycin resistance in the T₂ generation indicated 1 to 3 integration events. GUS expression and RT-PCR of the ipt gene confirmed the senescence-specificity of the SAG12 promoter. Morphologically, the transgenic lines appeared healthy and normal. Transgenic plants began to flower at the same time as wild-type plants, but the period from flowering to senescence was lengthened by 7 to 12 days. Tolerance of the transgenic plants to waterlogging and complete submergence was assayed in three independent experiments. All four transgenic lines were consistently more tolerant to flooding than wild-type plants. The results indicated that endogenously produced cytokinin can regulate senescence caused by flooding stress, thereby, increasing plant tolerance to flooding. This study provides a novel mechanism to improve flooding tolerance in plants.     

Morphological alterations by ectopic expression of the rice OsMADS4 gene in tobacco plants

OsMADS4, a rice MADS-box gene, is a member of the GLO/PI family that specifies the identity of petals and stamens in combination with other MADS-box genes. We report here the ectopic expression of OsMADS4 fused to the CaMV 35S promoter in tobacco plants. Transgenic plants carrying the CaMV 35S promoter::OsMADS4 construct generated mutant flowers with a mosaic carpel, in which the tissue around the nectary was elongated and the styles reduced. The fruits were distorted, but viable seeds did develop. These phenotypes mimicked those of transgenic tobacco plants that ectopically express Antirrhinum GLO. However, unlike GLO, OsMADS4 did not cause any homeotic change in the first whorl of the transgenic flowers. These results suggest that the functional role of OsMADS4 in the outer whorls has diverged from that of its dicot counterparts.