Activation tagging in plants: a tool for gene discovery

A significant limitation of classical loss-of-function screens designed to dissect genetic pathways is that they rarely uncover genes that function redundantly, are compensated by alternative metabolic or regulatory circuits, or which have an additional role in early embryo or gametophyte development. Activation T-DNA tagging is one approach that has emerged in plants to help circumvent these potential problems. This technique utilises a T-DNA sequence that contains four tandem copies of the cauliflower mosaic virus (CaMV) 35S enhancer sequence. This element enhances the expression of neighbouring genes either side of the randomly integrated T-DNA tag, resulting in gain-of-function phenotypes. Activation tagging has identified a number of genes fundamental to plant development, metabolism and disease resistance in Arabidopsis. This review provides selected examples of these discoveries to highlight the utility of this technology. The recent development of activation tagging strategies for other model plant systems and the construction of new more sophisticated vectors for the generation of conditional alleles are also discussed. These recent advances have significantly expanded the horizons for gain-of-function genetics in plants.     

Activation tagging: a means of isolating genes implicated as playing a role in plant growth and development

Activation T-DNA tagging has been used to generate a variety of tobacco cell lines selected by their ability to grow either in the absence of auxin or cytokinin in the culture media, or under selective levels of an inhibitor of polyamine biosynthesis. The majority of the cell lines studied in detail contain single T-DNA inserts genetically co-segregating with the selected phenotype. While most of the plants regenerated from the mutant cell lines appear phenotypically normal, several display phenotypes which could be inferred to result from disturbances in the content, or the metabolism, of auxins and cytokinins, or polyamines. The tagging vector is designed to allow the isolation of tagged plant genes by plasmid rescue. Confirmation that the genomic sequence responsible for the selected phenotype has indeed isolated is provided by PEG-mediated protoplast DNA uptake of rescued plasmids followed by selection for protoplast growth under the original selective conditions. Several plasmids have been rescued from the mutant lines which confer on transfected protoplasts the ability to grow either in the absence of auxin or cytokinin in the culture media, or under selective levels of an inhibitor of polyamine biosynthesis. This review describes the background to activation tagging and our progress in characterizing the genes that have been tagged in the mutant lines we have generated.     

Activation tagging,a novel tool to dissect the functions of a gene family

In a screen for morphological mutants from the T1 generation of approximately 50 000 activation-tagging lines, we isolated four dominant mutants that showed hyponastic leaves, downward-pointing flowers and decreased apical dominance. We designated them isoginchaku (iso). The iso-1D and iso-2D are allelic mutants caused by activation of the AS2 gene. The T-DNAs were inserted in the 3' downstream region of AS2. Iso-3D and iso-4D are the other allelic mutants caused by activation of the ASL1/LBD36 gene. These two genes belong to the AS2 family that is composed of 42 genes in Arabidopsis. The only recessive mutation isolated from this gene family was of AS2, which resulted in a leaf morphology mutant. Applying reverse genetics using a database of activation-tagged T-DNA flanking sequences, we found a dominant mutant that we designated peacock1-D (pck1-D) in which the ASL5/LBD12 gene was activated by a T-DNA. The pck1-D mutants have lost apical dominance, have epinastic leaves and are sterile. These results strongly suggest that activation tagging is a powerful mutant-mining tool especially for genes that make up a gene family.     

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.     

Crosstalk between auxin, cytokinins, and sugars in the plant cell cycle

Plant meristems are utilization sinks, in which cell division activity governs sink strength. However, the molecular mechanisms by which cell division activity and sink strength are adjusted to a plant's developmental program in its environmental setting are not well understood. Mitogenic hormonal as well as metabolic signals drive and modulate the cell cycle, but a coherent idea of how this is accomplished, is still missing. Auxin and cytokinins are known as endogenous mitogens whose concentrations and timing, however, can be externally affected. Although the sites and mechanisms of signal interaction in cell cycle control have not yet been unravelled, crosstalk of sugar and phytohormone signals could be localized to several biochemical levels. At the expression level of cell cycle control genes, like cyclins, Cdks, and others, synergistic but also antagonistic interactions could be demonstrated. Another level of crosstalk is that of signal generation or modulation. Cytokinins affect the activity of extracellular invertases and hexose-uptake carriers and thus impinge on an intracellular sugar signal. With tobacco BY-2 cells, a coordinated control of cell cycle activity at both regulatory levels could be shown. Comparison of the results obtained with the root cell-representing BY-2 cells with literature data from shoot tissues or green cell cultures of Arabidopsis and Chenopodium suggests opposed and tissue-specific regulatory patterns of mitogenic signals and signal crosstalk in root and shoot meristems.     

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     

激活标签法及其在植物基因工程上的应用

激活标签法是新近发展起来的一种用于基因分离和鉴定的方法。它通过诱变使特定内源基因发生过量表达,而产生显性功能获得型突变,从而对基因进行鉴定和分析。因为具有独特的性质已使其成为发现新基因和进行基因功能分析的有效工具。本文综述了激活标签法的原理、研究现状和在植物基因工程上的应用。 Abstract:Activation tagging is a new method for isolation and functional identification.It can generate dominant gain-of-function mutants by overexpression of a particular endogenous gene.Due to this special characteristics of activation tagging,this method has been a powerful tool for new gene discovery and gene functional analysis.This paper reviewed the principle and study conditions of activation tagging,as well as its use in plant genetic engineering.     

Flower-specific expression of the Agrobacterium tumefaciens isopentenyltransferase gene results in radial expansion of floral organs in Petunia hybrida

Biotechnology has the potential to modify commercially important traits of crops, such as fruit size and stress tolerance. To date, the floricultural industry has not profited significantly from these possibilities to manipulate, for example, flower size. Cytokinins are known to be involved in many aspects of plant development, including cell division. Increasing the amount of cytokinins has the potential to increase the size of an organ, such as the flower or the fruit. The Agrobacterium tumefaciens cytokinin biosynthesis gene isopentenyltransferase ( ipt ) has been shown to increase cytokinin levels when introduced into plants. Moreover, it has a dramatic effect on the vegetative development of plants. The expression of the ipt gene under the control of the flower-specific Arabidopsis APETALA3 promoter in petunia ( Petunia hybrida ) increases the flower size dramatically, but with no effect on vegetative development. The resulting transgenic plants produced flowers with larger corolla diameter and greater total floral fresh weight. This strategy has the potential for use in the production of ornamental crops with large flowers and crop species with larger fruit.     

Quantification of the export of cytokinins from roots to shoots of Rosa hybrida and their degradation rate in the shoot

Cytokinins from the roots may be involved in regulating rose ( Rosa hybrida ) shoot growth and development. The objective of this study was to estimate the export of cytokinins from the roots and their degradation rate in the shoot, which were expected to be correlated with plant development. Hence, the total cytokinin content of the shoot, the concentration of zeatin riboside (ZR) in bleeding sap, and the transpiration rates in three stages of development were determined. The estimations performed are based on the assumption that the cytokinin concentration in bleeding sap is representative for the cytokinin concentration in xylem sap in situ. This was verified by comparing the ZR concentration in bleeding sap and in sap obtaíned after pressurizing the root system to a level equivalent to the leaf water potential; no significant differences could be found. The import of cytokinins could not be correlated with plant development, as it increased linearly with time. The estimated relative degradation rate of cytokinins in the shoot decreased as the plants matured. The half-life of cytokinins in the shoot was found to be approximately 1 day, indicating that cytokinins are rapidly metabolized in the shoot.     

RFLP tagging of a salt tolerance gene in rice

A salt tolerant rice mutant (M-20) was obtained through selection in vitro. Its tolerance was stably inherited over eight generations and most traints between M-20 and its sensitive original 77–170 (Oryza sativa) were very similar. By deriving an F₂ population of M-20 × 77–170 and splitting every F₂ individual into two parts, with one part planted in normal conditions and another part in saline conditions, the inheritance of salt tolerance in rice was studied. Under normal conditions, there was no apparent segregation among F₂ individuals. Under saline conditions, however, the segregation of traits was obvious. According to our standards, the ratio of salt sensitive:moderately-tolerant:tolerant plants was 25:42:18, in accordance with a 1:2:1 ratio. It suggested that the improvement of salt tolerance in our materials was induced by the mutation of a major tolerant gene which showed incomplete dominance. By use of 130 RFLP probes distributed throughout the rice genome, the gene was tagged by a single copy DNA probe, RG4, which was located on chromosome 7. The genetic distance between the salt tolerant gene and RG4 was 7.0 ± 2.9 cM. Based on the split method, a method which could be currently used to evaluate the damage of salt stress in rice was proposed.     

Somatic mobility of the maize element Ac and its utility for gene tagging in aspen

We have investigated the somatic activity of the maize Activator (Ac) element in aspen with the objective of developing an efficient transposon-based system for gene isolation in a model tree species. The analysis of the new insertion sites revealed the exact reconstitution of the Ac, however, aberrant transposition events were also found. Characterization of the genomic sequences flanking the Ac insertions showed that about one third (22/75) of the sequences were significantly similar to sequences represented in public databases and might correspond to genes. The frequency of Ac landing into coding regions was about two-fold higher when compared to the frequency of T-DNA hitting the predicted genes (5/32) in the aspen genome. Thus, Ac is demonstrated to be a potentially useful heterologous transposon tag in a tree species. This is the first report on transposon-based gene tagging in a tree species describing the excision and reinsertion of transposable element into new genomic positions. We also suggest a heterologous transposon tagging strategy that can be used in aspen somatic cells to obtain dominant gain-of-function mutants and recessive loss-of-function mutants overcoming the regeneration time barrier of a long-lived tree species.     

Analysis of insecticidal activity in transgenic plants carrying the ipt plant growth hormone gene

The expression of a bacterial cytokinin biosynthesis gene (PI-II-ipt) in Nicotiana plumbaginifolia Viviani plants has been correlated with enhanced resistance to Manduca sexta and Myzus persicae. We expressed the PI-II-ipt gene in N. tabacum and Lycopersicon esculentum and observed similar antifeedent effects with the transgenic tobacco but not tomato. A 30 to 50 % reduction in larval weight gain was observed with some of the tomato plants but these results could not be repeated consistently. Leaf surface extracts from transgenic N. plumbaginifolia leaves killed 100 % of M. sexta second instars at concentrations of 0.05 % (w/v) whereas the N. tabacum extracts were at least 20 times less active. Extract suspensions were stable for up to 2 days at ambient temperatures below 42 °C and for at least 3 months at 4 °C when stored in the dark. HPLC analysis of the N. plumbaginifolia extracts yielded an active fraction that reduced hatching of M. sexta eggs by 30 % and killed first, second and third instars within 24, 48 and 72 hours of exposure, respectively. The activity appears to be associated with oxygen-containing aliphatic compounds, possibly diterpenes, as analyzed by TLC, UV absorption and fragmentation with EIMS. Based on the partial characterization of this activity, the production, secretion or accumulation of secondary metabolites in leaves of cytokinin producing PI-II-ipt N. plumbagini-folia plants appears to be responsible for the observed insect resistance.     

Regulation of gene action in Petunia hybrida: Unstable alleles of a gene for flower colour

Summary In a progeny of a selfed individual of the dark red-flowered cultivar Roter Vogel some white-flowered plants appeared as the result of a mutation of the genetic factor Anl involved in anthocyanin synthesis. The white flowers of these plants had red spots owing to back-mutations in the dermal cells of the young corolla.Owing to a striking unstability of the new allele of Anl, a number of mutants originated which differ mutually in the frequency of reversion, which expressed itself in the very substantial differences in the spot density of the limb of the corolla. Between a mean number of less than one spot per cm² of the limb and a mean number of over 10.000 spots/cm², a series of transitions was found.The reversions did not remain restricted to the young epidermis but also occurred in sporogenous tissues. This resulted in the appearance of selfcoloured red descendants of plants with red-spotted white flowers. There is a positive correlation between the spot density of the parent plants and the percentage of plants with completely red corollas.The red spots on the corolla usually have the same colour as the wild type (Roter Vogel), but occasionally mutants occur with paler spots, the colour varying from a very pale pink to a red nearly as deep as in the wild type. The selfcoloured descendants of such mutants also show this colour variation from pale pink to red.On the grounds of these observations a theory was formulated which postulates that the Anl locus consists of a structural gene responsible for an enzyme active during anthocyanin synthesis and a regulatory element built up from intermediate repetitive DNA. This regulatory element in turn is built up of two components, one of which, the mutator, decides the activation of the structural gene while the other, the expressor, modifies the rate of activation. The mutations must be considered representative of larger or smaller deletions within one or both of these components. Reversions are the result of the restoration of the deletions by means of an amplification of the repetitive DNA in dividing cells of the developing flower buds.     

Phenylurea cytokinins assayed for induction of shoot buds in the moss Funaria hygrometrica

The induction of shoot buds from the filamentous protonema of moss is a classic bioassay for cytokinin. While a large literature documents this response in many species of moss and for a wide range of natural and synthetic cytokinins, to date only substituted adenine cytokinins have been examined in detail. This paper shows that at least some of the novel phenylurea cytokinins will induce bud formation in mosses. Funaria responds to thidiazuron much as it responds to benzyladenine. Exposure to either substance results in log-linear dose-dependent increases in bud number that reach similar maximal numbers of buds at the optimal concentration of compound. The related compound chloro-pyridyl-phenylurea (CPPU) is slightly less active, but induces buds over a wider range of concentration. Carbanilide (diphenylurea or DPU), an active cytokinin in other systems, induces very few buds in Funaria, but does so over a wide range of concentration. Bioassay of mixtures of benzyladenine and DPU finds no evidence of competition for cytokinin receptors. That result could support suggestions that the phenylurea cytokinins act indirectly, by altering endogenous cytokinin metabolism, but we favor another interpretation. Unlike other cytokinin-responsive systems, the induction of buds from moss protonema involves two cytokinin-mediated events. The number of buds is controlled by the second cytokinin-mediated event. If DPU has little or no affinity for the receptor triggering this second event, DPU treatments will produce few to no buds, and kinetic analysis using bud number would find no evidence for competition with benzyladenine. Our results would support the hypothesis that bud induction in Funaria involves two chemically distinct cytokinin receptors.     

Extended flower longevity of Petunia hybrida plants transformed with boers,a mutated ERS gene of Brassica oleracea

Petunia x hybridaHort.Vilm.-Andr. was transformed with boers, a mutatedallele of BOERS, an ethylene receptor sensor gene ofBrassica oleracea.boers was obtained by removing anEcoRI cutting site with a silent mutation at Gly-521 andintroducing a point mutation at Ile-62, replacing isoleucine withphenylalanine. Transformation was Agrobacterium tumefaciens mediated.Hygromycin resistant regenerants were tentatively confirmed as transformants byPCR's for HPH and boers and moredefinitively by Southern hybridization of genomic DNA with pBOERS4421. Flowersof transgenic plants retained turgidity and pigmentation considerably longerthan those of untransformed controls, whether left undisturbed on plants orexcised and placed in water. Furthermore, flowers were unaffected by exposureto exogenous ethylene. Excised shoots of transgenic plants released considerablymore ethylene than those of untransformed plants. Transformed plants alsoproduced apparently larger flowers. Unexpectedly higher mortality was observed,suggesting that the ethylene insensitive petunia plants were also lower indisease resistance.     

Promoter activity of a putative pollen monosaccharide transporter in Petunia hybrida and characterisation of a transposon insertion mutant

Summary. For the growth of the male reproductive cells of plants, the pollen, the presence of sufficient sucrose or monosaccharides is of vital importance. From Petunia hybrida a pollen-specific putative monosaccharide transporter designated PMT1 (for petunia monosaccharide transporter) has been identified previously. The present work provides an in-depth analysis and characterisation of PMT1 in the context of pollen development with the GUS reporter gene and an insertion mutant. The promoter of the pollen-specific putative PMT1 gene has been isolated by inverse PCR and sequenced. Analysis of plants transformed with the promoter-GUS fusion confirmed the specificity of this gene, belonging to the late pollen-specific expressed genes. GUS activity was detected even after 24 h of in vitro pollen germination, at the pollen tube tip. To elucidate the importance of PMT1 for gametophyte development and fertilisation, we isolated a mutant plant containing a transposon insertion in the PMT1 gene by the dTph1 transposon-tagging PCR-based assay. The PMT1 mutant contained a dTph1 insertion in position 1474 bp of the transcribing part of the gene, before the last two transmembrane-spanning domains. Analysis of the progeny of the heterozygous mutant after selfing revealed no alterations in pollen viability and fertility. Mature pollen grains of a plant homozygous for the transposon insertion were able to germinate in vitro in a medium containing sucrose, glucose, or fructose, which indicates that PMT1 is not essential for pollen survival. Several explanations for these results are discussed in the present work. Correspondence and reprints (present address): Department of Plant Biology, University of Granada. Fuentenueva s/n, 18001 Granada, Spain. Present address: Swammerdam Institute for Life Sciences, Amsterdam, the Netherlands.