New insights into the biology of cytokinin degradation.

A survey of recent results is presented concerning the role of cytokinin degradation in plants, which is catalyzed by cytokinin oxidase/dehydrogenase (CKX) enzymes. An overview of Arabidopsis CKX gene expression suggests that their differential regulation by biotic and abiotic factors contributes significantly to functional specification. Here, we show using reporter gene and semiquantitative RT-PCR analyses regulation of individual CKX genes by cytokinin, auxin, ABA, and phosphate starvation. Partially overlapping expression domains of CKX genes and cytokinin-synthesizing IPT genes in meristematic tissues and endo-reduplicating cells lend support for a locally restricted function of cytokinin. On the other hand, their expression in vascular tissue suggests a function in controlling transported cytokinin. Recent studies led to a model for the biochemical reaction mechanism of CKX-mediated catalysis, which was refined on the basis of the three-dimensional enzyme structure. Last but not least, the developmental functions of CKX enzymes are addressed. The recent identification of the rice OSCKX2 gene as an important novel breeding tool is highlighted. Together the results corroborate the relevance of metabolic control in determining cytokinin activity.

Regulation of plant growth by cytokinin.

Cytokinins are a class of plant-specific hormones that play a central role during the cell cycle and influence numerous developmental programs. Because of the lack of biosynthetic and signaling mutants, the regulatory roles of cytokinins are not well understood. We genetically engineered cytokinin oxidase expression in transgenic tobacco plants to reduce their endogenous cytokinin content. Cytokinin-deficient plants developed stunted shoots with smaller apical meristems. The plastochrone was prolonged, and leaf cell production was only 3-4% that of wild type, indicating an absolute requirement of cytokinins for leaf growth. In contrast, root meristems of transgenic plants were enlarged and gave rise to faster growing and more branched roots. These results suggest that cytokinins are an important regulatory factor of plant meristem activity and morphogenesis, with opposing roles in shoots and roots.

CREam of cytokinin signalling: receptor identified.

Cytokinins play a central role in the regulation of plant cell division and numerous developmental processes. Pleiotropic effects have made studies of this hormone difficult, and cytokinin signalling pathways have long remained elusive. The recent identification of CRE1 (a histidine kinase identical to AHK4 and WOL) as the cytokinin receptor of Arabidopsis thaliana is a landmark in cytokinin research. Mutations have been identified in CRE1, and the phenotype of loss-of-function mutations sheds new light on the role of cytokinins in plant development. This article describes the experimental paths leading to receptor identification and the current interpretation of its function.

The ORF8 gene product of Agrobacterium rhizogenes TL-DNA has tryptophan 2-monooxygenase activity.

The open reading frame 8 (ORF8) is located on the TL-DNA of the phytopathogenic soil bacterium Agrobacterium rhizogenes strain A4. The predicted ORF8 protein has a particular structure and is possibly a natural fusion protein. The N-terminal domain shows homology to the A. rhizogenes rolB protein and may modulate the auxin responsiveness of host cells. The C terminus has up to 38% homology to tryptophan 2-monooxygenases (t2m). We show that ORF8 overexpressing plants contain a fivefold higher concentration of indole-3-acetamide (IAM) than untransformed plants. Protein extracts from seedlings and Escherichia coli overexpressing ORF8 show significantly higher turnover rates of tryptophan to IAM than negative controls. We conclude that the ORF8 gene product has tryptophan 2-monooxygenase activity.

Hormone autotrophic growth and differentiation identifies mutant lines of Arabidopsis with altered cytokinin and auxin content or signaling.

We describe mutant tissue lines of Arabidopsis that are able to grow in vitro as callus on hormone-free medium. The 14 lines presented here show different hormone autotrophic differentiation behaviors that can be classified into three categories: (a) forming roots (rooty callus), (b) forming shoots or shoot-like structures (shooty callus), or (c) growing without organ formation (callus). Three fast-growing lines showed altered steady-state mRNA levels of the Cdc2 and CycD3 cell cycle genes. Three of the six rooty callus lines contained about 20- to 30-fold higher levels of auxins than wild-type callus. These and two other lines with normal auxin content showed an increased steady-state level of IAA1 and IAA2 transcripts in the absence of exogenous auxin. Five of the six shooty callus lines had increased steady-state mRNA levels of the CKI1 gene and/or of the homeobox genes KNAT1 and STM, suggesting that the phenotype is linked to altered cytokinin signaling. Also, one cytokinin-overproducing line with only 5% of wild-type cytokinin oxidase activity was identified. These results indicate that screening for hormone-autonomous growth identifies mutants with altered hormone content or signaling.

Increased steady state mRNA levels of the STM and KNAT1 homeobox genes in cytokinin overproducing Arabidopsis thaliana indicate a role for cytokinins in the shoot apical meristem.

This study investigates the consequences of endogenously enhanced biosynthesis of the plant hormone cytokinin in Arabidopsis thaliana (L.) Heynh. Transcriptional control of the bacterial ipt gene by the Drosophila melanogaster hsp70 promoter enabled temperature-dependent increased cytokinin production in transgenic plants. Heat-treated plants accumulated higher levels of unbound and bound zeatin-type cyto-kinins, the latter being preferentially N-conjugated glucosides. Cytokinin overproduction significantly increased the biomass of seedlings. Ipt transgenics had higher steady state mRNA levels of the shoot meristem specifying homeobox genes KNAT1 and STM, similar to the cytokinin-overproducing shoot meristem mutant amp1 (hpt, cop2, pt) This finding, together with previously described phenotypic similarities between transgenic cytokinin-overproducing plants and plants overexpressing the KNAT1 or KN1 genes, suggests that these factors act on the same pathway. We hypothesize that cytokinins act upstream of KNAT1 and STM. The influence of cytokinins on homeobox genes provides a link between the hormone and the developmental genes and indicates a role for cytokinins in the shoot apical meristem.

Gain of function assays identify non-rol genes from Agrobacterium rhizogenes TL-DNA that alter plant morphogenesis or hormone sensitivity.

This study tested the morphogenetic potential of 15 open reading frames of the TL-DNA of Agrobacterium rhizogenes strain HRI. These open reading frames were expressed individually under the control of the 35S RNA promoter in transgenic tobacco plants (Nicotiana tabacum L.). Expression of three T-DNA loci, ORF3n, ORF8 and ORF13, alters plant morphogenesis or the response of transgenic tissues to plant hormones. ORF3n transgenic plants are characterized by retarded flowering, altered internode elongation, altered leaf shape and, in particular, leaf tip necrosis. ORF3n and ORF8 expression reduces the sensitivity to auxin and cytokinin in combination or auxin alone. Tetracycline-dependent expression of ORF13 overcomes a selection of low levels of expression during plant regeneration and reveals a strong inhibitory effect of the ORF13 gene product on cell division and cell elongation. We conclude that the A. rhizogenes TL-DNA harbors genetic information that is important for pathogenicity apart from the well studied rol genes. We propose that these genes play mainly a negative regulatory role during pathogenesis. Moreover, these loci might be relevant to successful infections in specific host plants.

A putative rolB gene homologue of the Agrobacterium rhizogenes TR-DNA has different morphogenetic activity in tobacco than rolB.

Agrobacterium rhizogenes strains of the agropine type harbor on their Ri-plasmid two T-DNAs, a left TL-DNA and a right TR-DNA. The rolB gene of the TL-DNA is the major factor in the pathogenesis of the hairy-root disease and its constitutive expression interfere profoundly with plant morphogenesis. We have tested whether the expression of its sequence related putative homologue from the TR-DNA (rolBTR) may cause also bacterial virulence or affect plant development. Unlike rolB, rolBTR is unable to induce root formation on tobacco leaf discs. Tobacco plants expressing a chimeric 35S::rolBTR gene have reduced stature, off-shoots at the stem base and bent and wrinkled leaves with epinastic growth. 14 N-terminal amino acids which are absent in the rolB protein are indispensable to rolBTR protein activity. The characteristic tyrosine phosphatase super family motif CX5R is absent in the rolBTR protein. For rolB this motif is possibly functionally relevant. We conclude that the rolBTR gene product has morphogenic activity but is not a functional homologue of the rolB protein.

Morphology and tuber formation of in-vitro-grown potato plants harboring the yeast invertase gene and/or the rolC gene.

Growth and tuber formation of transgenic potato plants (Solanum tuberosum cv. Désirée) harboring the yeast invertase gene and the rolC gene individually or in combination under the transcriptional control of the patatin promoter were investigated under different conditions in vitro. Plants expressing only the invertase gene were morphologically similar to control plants. rolC transgenic plants had an increased tiller number, improved root growth, and a higher total biomass. Tuber formation and growth were altered by the introduced transgenes. The sucrose requirement to induce tubers was shifted to lower or higher concentrations for invertase- or rolC-expressing clones, respectively. In addition, rolC plants formed tubers of altered morphology. A comparison with soil-grown plants showed that morphological parameters can be predicted to some extent from in vitro studies, while for reliable prescreening of parameters concerning tuber formation and growth, an optimization of currently used protocols is necessary.

Conditional transgenic expression of the ipt gene indicates a function for cytokinins in paracrine signaling in whole tobacco plants.

This study investigated whether an increased production of the plant hormone cytokinin in roots, the main site of its synthesis and putative signaling organ, can influence developmental events, such as growth of axillary shoot meristems or leaf senescence, in the plant shoot. To this end, transgenic tobacco plants (Nicotiana tabacum L.) were generated that conditionally overproduce cytokinins. These plants harbour the ipt gene under the transcriptional control of a modified 35S promoter that is repressed in plants with high titers of tetracycline repressor protein. De-repression of transcription led to a rapid more than 50-fold increase of hormone concentration. The time course of changes in the steady-state levels of 16 different cytokinin metabolites, as a consequence of IPT enzyme activity, was monitored in different plant tissues. Zeatin riboside was the first and most dramatically increased product; zeatin, dihydrozeatin and glucosides accumulated later. The consequences of enhanced cytokinin synthesis remained mainly restricted to the site of hormone production. For example, de-repression of ipt gene transcription in lateral buds caused the growth of single buds only at the site of tetracycline application. In reciprocal grafts of transgenic plants with wild-type plants, no biological cytokinin effects, i.e. growth of lateral shoot meristems or sequential leaf senescence, were observed in the non-transgenic plant part. Also, the increase in steady-state levels of cytokinins remained restricted mainly to the transgenic part, despite a specific increase of the zeatin riboside concentration in the transpiration stream. These results question the role of cytokinins as a long-range root-to-shoot signal in correlative control of apical dominance and sequential leaf senescence of tobacco, and support the assumption that this hormone is relevant to paracrine signaling.

Changes in Cytokinin Content and Cytokinin Oxidase Activity in Response to Derepression of ipt Gene Transcription in Transgenic Tobacco Calli and Plants.

Metabolic control of cytokinin oxidase by its substrate was investigated in planta using wild-type (WT) and conditionally ipt gene-expressing transgenic (IPT) tobacco (Nicotiana tabacum L.) callus cultures and plants. The derepression of the tetracycline (Tc)-dependent ipt gene transcription was followed by a progressive, more than 100-fold increase in total cytokinin content in IPT calli. The activity of cytokinin oxidase extracted from these calli began to increase 16 to 20 h after gene derepression, and after 13 d it was 10-fold higher than from Tc-treated WT calli. An increase in cytokinin oxidase activity, as a consequence of elevated cytokinin levels, was found in detached leaves (8-fold after 4 d) and in roots of intact plants (4-fold after 3 d). The partially purified cytokinin oxidase from WT, repressed IPT, and Tc-derepressed IPT tobacco calli exhibited similar characteristics. It had the same broad pH optimum (pH 6.5-8.5), its activity in vitro was enhanced 4-fold in the presence of copper-imidazole, and the apparent Km(N6-[[delta]2iso-pentenyl]adenine) values were in the range of 3.1 to 4.9 [mu]M. The increase in cytokinin oxidase activity in cytokinin-overproducing tissue was associated with the accumulation of a glycosylated form of the enzyme. The present data indicate the substrate induction of cytokinin oxidase activity in different tobacco tissues, which may contribute to hormone homeostasis.

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.

Gene silencing in transgenic tobacco hybrids: frequency of the event and visualization of somatic inactivation pattern.

We have investigated the stability of the expression of different T-DNA-borne genes in hybrid tobacco lines. These lines were constructed to rescue rolC-induced male sterility in kanamycin-resistant P35s-rolC transgenic tobacco plants by expression of rolC antisense genes. Using five different tester lines, a total of 158 hybrids was obtained. We observed inactivation of transgene expression in 20% of the F1 progeny and in 35% of the backcrossed F2 progeny, as indicated by the loss of kanamycin resistance. In 3% of all crosses complete loss of antibiotic resistance was noted, while in most affected hybrid progeny only part of the population became kanamycin sensitive. Single genes could be selectively inactivated on T-DNAs harboring several genes. Gene inactivation was not restricted to one of the two T-DNAs examined. Somatic silencing, visualized by a cell-specific 35SGUSINT marker gene, occurred in a random fashion or exhibited an inherited specific pattern. The type of somatic silencing pattern observed indicated developmental control of the process. Two phenotypic classes could be distinguished with respect to frequency and timing of the inactivation process. Rapid gene inactivation, occurring within a few weeks after germination of hybrid seedlings, was characterized by complete methylation of restriction sites in the promoter of the silenced gene, resetting of gene expression during meiosis, heredity of the developmentally controlled program of gene silencing in subsequent generations, and rapid reactivation of gene expression after genetic separation of the different T-DNAs. In contrast, a slow type of gene inactivation was of a more stochastic nature and was recognized only in hybrids of the backcrossed F2 generation. In this case the degree of promoter methylation, which could extend beyond the T-DNA borders, was not correlated with the reduction in steady-state poly(A)+ mRNA levels, the silenced state was transmitted through meiosis and reactivation lasted several generations. The implications of the observations for our understanding of the gene inactivation process are discussed.

Promoter tagging with a promoterless ipt gene leads to cytokinin-induced phenotypic variability in transgenic tobacco plants:implications of gene dosage effects.

Tobacco plants have been transformed with a T-DNA construct harboring a promoterless cytokinin-synthesizing ipt gene close to the right T-DNA border. Eighteen out of 85 transgenic clones displayed phenotypic alternations typical for an enhanced cytokinin production. Northern blot analysis confirmed the transcriptional activation of the introduced gene by tagged plant promoters. The concentration of cytokinins, expressed as zeatinriboside equivalents, was increased up to sevenfold in transgenic tissues. These increases in cytokinin levels resulted in major developmental changes. Transgenic clones exhibited to different levels traits of a general cytokinin-syndrome, i.e. reduced root growth, reduced apical dominance, reduced leaf surface, reduced growth of the stem and retarded leaf senescence or displayed localized and developmentally specific cytokinin-induced alterations in otherwise normally developing plants. These traits were in particular a simultaneous break of dormancy in all axillary buds before or at the onset of flowering or the reorientation of the developmental pathway of secondary meristems or terminally differentiated cells. This indicates that endogenously produced cytokinins not only influence different growth parameters but have the potential to alter differentiation pattern. The results show that stably inherited developmental alterations due to a general or localized cytokinin overproduction can be obtained by the promoter-tagging approach. The investigation of gene dosage effects in homozygote plants readdresses the question of threshold levels for cytokinin effects on the developmental program of plants.

Efficiency of the tetracycline-dependent gene expression system: complete suppression and efficient induction of the rolB phenotype in transgenic plants.

We have investigated the use of the tetracycline-dependent gene expression system to regenerate and propagate tobacco plants transformed with a gene whose product--when highly expressed--interferes with regeneration and/or further reproduction. Plants transformed with the Agrobacterium rhizogenes rolB gene under the control of the tetracycline-dependent expression system were phenotypically indistinguishable from wild type owing to efficient repression of the promoter. Induction of the rolB gene with tetracycline led to high-level expression of the rolB mRNA, which resulted in extremely stunted plants with necrotic and wrinkled leaves that did not develop a floral meristem. Upon cessation of tetracycline treatment healthy shoots developed even from severely affected meristems. Data on the dose response of the rolB phenotype as a function of tetracycline concentration demonstrate that the tetracycline-dependent gene expression system can be used to modulate the manifestation of a particular phenotype.

Phenotype and hormonal status of transgenic tobacco plants overexpressing the rolA gene of Agrobacterium rhizogenes T-DNA.

The rolA gene of the TL-DNA of Agrobacterium rhizogenes Ri-plasmid plays a major role in establishing the hairy root syndrome in transgenic plants. Transgenic tobacco plants (Nicotiana tabacum L.) expressing constitutively the rolA gene under the transcriptional control of the 35S RNA promoter show pronounced phenotypical alterations. P35S-rolA transgenic tobacco plants are characterized by stunted growth, dark green wrinkled leaves with an altered length-to-width ratio, condensed influorescences, retarded onset of flowering, a reduced number of flowers and shortened styles. To investigate whether the pleiotropic alterations of growth and development are linked to an altered hormonal status we have compared the immunoreactive content of indole-3-acetic acid, cytokinins, abscisic acid, gibberellin and the ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC) of seedlings and different tissues of P35S-rolA transgenic plants, transgenic plants expressing the rolA gene under control of its own phloem-specific promoter and wild-type plants. Multiple tissue-specific alterations of phytohormone concentrations are the consequence of rolA gene activity. Changes of phytohormonal content can explain part of the rolA-induced phenotypic alterations. Most strikingly, in young and fully developed leaves of rolA and P35S-rolA transgenic clones a 40-60% reduction of immunoreactive gibberellin A1 was found, as compared to wild-type leaves. Treatment of wild-type tobacco plants with inhibitors of gibberellin biosynthesis phenotypic alterations similar to those of rolA transgenic plants. This suggests that the reduction of gibberellic acid content is indirectly but causally involved in rolA-induced alterations of stem elongation and planar leaf blade growth.

Constitutive or light-regulated expression of the rolC gene in transgenic potato plants has different effects on yield attributes and tuber carbohydrate composition.

Tetraploid potato clones, transgenic for the rolC gene of Agrobacterium rhizogenes under control of the light-inducible ribulose bisphosphate carboxylase small subunit promoter (rbcS-rolC), were compared, with respect to yield attributes and tuber carbohydrates, with transformed and untransformed controls and with 35S-rolC transgenic potato plants. In rbcS-rolC plants, the expression of the rolC gene was located mainly in leaves, while in 35S-rolC plant transcripts were detected as well in shoots and roots. Phenotypically, rbcS-rolC transgenic plants were found to be slightly reduced in plant size with a few more tillers than control plants. Photosynthetic rate and chlorophyll content were significantly lower in all rolC transgenic plants irrespective of the type of construct used. Tuber yield was not significantly different between controls and rbcS-rolC transgenic plants, but was reduced in the 35S-rolC transformants. Sucrose level was unchanged in all rolC clones investigated, whereas fructose content was significantly enhanced in 35S-rolC transformants, but not in the plants expressing the rolC gene in aerial plant parts only. In both types of rolC transgenic plants, glucose content was lower than in controls, resulting in a significant reduction of reducing sugar in tubers. The results suggest a hormonal influence on the carbohydrate composition of potato tubers.

Restoration of fertility by antisense RNA in genetically engineered male sterile tobacco plants.

Transgenic tobacco plants (Nicotiana tabacum L.) expressing the rolC gene of Agrobacterium rhizogenes under the transcriptional control of the 35S RNA promoter are male sterile. When these plants are genetically crossed with others containing the rolC gene linked in antisense orientation to the 35S RNA promoter, hybrid progeny display restoration of male fertility. Moreover, hybrid progeny are revertant for other features of the rolC phenotype, such as restoration of plant height, leaf pigment content and female fertility. The level of restoration of the characteristics of untransformed tobacco appeared to be independent of the steady-state level of antisense RNA. Addition of six transcriptional enhancer sequences upstream of the 35S transcriptional start region in the antisense construct led to a higher steady-state level of antisense RNA than that produced using a promoter linked to a single enhancer sequence. However no significant difference was observed in the level of attenuation of the rolC phenotype in the progeny of crosses with either one or six transcriptional enhancers linked to the antisense rolC gene. Antisense constructs comprising only 189 bp of the rolC 5' coding region appeared less efficient in attenuating the rolC phenotype than those including the whole rolC coding region as well as its 3' untranslated region. Furthermore, results from experiments on light-controlled rolC gene expression indicate that microsporogenesis is sensitive to rolC gene action during the early stages of flower development.

Transgenic tobacco plants regenerated from leaf disks can be periclinal chimeras.

Amongst rolC transgenic tobacco plants regenerated from leaf disks 6.5% are periclinal chimeras, i.e. plants with genetically different cell populations in different cell layers. The expression of the rolC gene of Agrobacterium rhizogenes causes a reduction in pigment content in leaves. The chimeric composition of the regenerated plants becomes thus apparent as light green leaf tissue in the transgenic region, tissue flanked by dark green wild-type sectors. Southern and northern blot analysis confirmed the chimeric nature of such plants. Investigation of selfed progeny of chimeric plants on selective media indicates that layer invasion in reproductive tissues can occur in tobacco early during the formation of the flower buds. The results show (1) that tobacco plants regenerated from leaf disks and grown on selective media have not necessarily the same clonal origin and (2) that they can give rise to non-transgenic offspring. The chimeric plants provide insight on the effect of rolC gene expression on microsporogenesis.