Iris Heidmann

Epigenetic changes in the expression of the maize A1 gene in Petunia hybrida: role of numbers of integrated gene copies and state of methylation.

SummaryThePetunia hybrida mutant RL01 is white flowering due to a genetic block in the anthocyanin pathway. The introduction of the maize Al cDNA under the control of the CaMV 35S RNA promoter leads to the production of pelargonidin derivatives, resulting in a brick red flower phenotype. Among the transgenic petunia plants the pigmentation of the petals exhibited different expression patterns which could be categorized into the ‘red’, the ‘variegated’, and the ‘white’ phenotype. This system proved to be especially suitable for the investigation of gene expression by simply looking at the pigmentation pattern of the petals. The uniformity of floral pelargonidin pigmentation is inversely correlated with the number of integrated Al copies. Furthermore, a correlation was found between the methylation status of the 35S RNA promoter and the instability of the floral pelargonidin coloration. The status of promoter methylation controlling the expression of the Al gene seems to be influenced by the copy number and the chromosomal position of the transferred gene.

Endogenous and environmental factors influence 35S promoter methylation of a maize A1 gene construct in transgenic petunia and its colour phenotype

Summary30000 transgenic petunia plants carrying a single copy of the maize A1 gene, encoding a dihydroflavonol reductase, which confers a salmon red flower colour phenotype on the petunia plant, were grown in a field test. During the growing season plants with flowers deviating from this salmon red colour, such as those showing white or variegated phenotypes and plants with flowers exhibiting only weak pigmentation were observed with varying frequencies. While four white flowering plants were shown at the molecular level to be mutants in which part of the A1 gene had been deleted, other white flowering plants, as well as 13 representative plants tested out of a total of 57 variegated individuals were not mutants but rather showed hypermethylation of the 35S promoter directing A1 gene expression. This was in contrast to the homogeneous fully red flowering plants in which no methylation of the 35S promoter was observed. While blossoms on plants flowering early in the season were predominantly red, later flowers on the same plants showed weaker coloration. Once again the reduction of the A1-specific phenotype correlated with the methylation of the 35S promoter. This variation in coloration seems to be dependent not only on exogenous but also on endogenous factors such as the age of the parental plant from which the seed was derived or the time at which crosses were made.

Heterologous expression of the BABY BOOM AP2/ERF transcription factor enhances the regeneration capacity of tobacco (Nicotiana tabacum L.)

Gain-of-function studies have shown that ectopic expression of the BABY BOOM (BBM) AP2/ERF domain transcription factor is sufficient to induce spontaneous somatic embryogenesis in Arabidopsis (Arabidopsis thaliana (L.) Heynh) and Brassica napus (B. napus L.) seedlings. Here we examined the effect of ectopic BBM expression on the development and regenerative capacity of tobacco (Nicotiana tabacum L.) through heterologous expression of Arabidopsis and B. napus BBM genes. 35S::BBM tobacco lines exhibited a number of the phenotypes previously observed in 35S::BBM Arabidopsis and B. napus transgenics, including callus formation, leaf rumpling, and sterility, but they did not undergo spontaneous somatic embryogenesis. 35S::BBM plants with severe ectopic expression phenotypes could not be assessed for enhanced regeneration at the seedling stage due to complete male and female sterility of the primary transformants, therefore fertile BBM ectopic expression lines with strong misexpression phenotypes were generated by expressing a steroid-inducible, post-translationally controlled BBM fusion protein (BBM:GR) under the control of a 35S promoter. These lines exhibited spontaneous shoot and root formation, while somatic embryogenesis could be induced from in-vitro germinated seedling hypocotyls cultured on media supplemented with cytokinin. Together these results suggest that ectopic BBM expression in transgenic tobacco also activates cell proliferation pathways, but differences exist between Arabidopsis/B. napus and N. tabacum with respect to their competence to respond to the BBM signalling molecule.

Efficient sweet pepper transformation mediated by the BABY BOOM transcription factor

Pepper (Capsicum L.) is a nutritionally and economically important crop that is cultivated throughout the world as a vegetable, condiment, and food additive. Genetic transformation using Agrobacterium tumefaciens (agrobacterium) is a powerful biotechnology tool that could be used in pepper to develop community-based functional genomics resources and to introduce important agronomic traits. However, pepper is considered to be highly recalcitrant for agrobacterium-mediated transformation, and current transformation protocols are either inefficient, cumbersome or highly genotype dependent. The main bottleneck in pepper transformation is the inability to generate cells that are competent for both regeneration and transformation. Here, we report that ectopic expression of the Brassica napus BABY BOOM AP2/ERF transcription factor overcomes this bottleneck and can be used to efficiently regenerate transgenic plants from otherwise recalcitrant sweet pepper (C. annuum) varieties. Transient activation of BABY BOOM in the progeny plants induced prolific cell regeneration and was used to produce a large number of somatic embryos that could be converted readily to seedlings. The data highlight the utility of combining biotechnology and classical plant tissue culture approaches to develop an efficient transformation and regeneration system for a highly recalcitrant vegetable crop.

Functional conservation and maintenance of expression pattern of FIDDLEHEAD-like genes in Arabidopsis and Antirrhinum

In Arabidopsis, loss of function of the epidermis-specific FDH gene coding for a putative β-ketoacyl-CoA synthase results in ectopic organ fusions in mutants. Corresponding mutants are not available for Antirrhinum majus, however, organ fusions can be induced in both species by chloroacetamide inhibitors of β-ketoacyl-CoA synthases using a chemical genetics approach. We isolated the ortholog of FDH from Antirrhinum majus, the ANTIRRHINUM FIDDLEHEAD (AFI ) gene, and showed that AFI complements fdh when expressed in the epidermis under control of the FDH promoter. Like FDH, the AFI gene exhibits protodermis- and epidermis-specific expression, and its promoter directs the expression of reporter genes to the epidermis in transgenic Antirrhinum and Arabidopsis. We demonstrate down-regulation of the FDH promoter in the epidermis of the ovary septum, thereby supporting the assumption that FDH-like genes may directly facilitate the cell–cell interactions that need to occur during carpel fusion and pollen tube growth. Up-regulation of FDH in the stomium, on the other hand, provides evidence for its possible involvement in cell separation during anther dehiscence. Down-regulation of the FDH and AFI promoters in the septum is observed in transgenic Arabidopsis but not in Antirrhinum plants. This probably reflects differences in the ontogeny of the ovary septum between the two species. We also show that epidermis-specific FDH-like genes may not be able to efficiently elongate fatty acid chains when misexpressed in seeds.

The use of African cassava mosaic virus as a vector system for plants

This paper describes the development of a gene-displacement vector based on DNA1, one of two single stranded circular genomic components of a bipartite geminivirus, African cassava mosaic virus (ACMV). The DNA1 molecules of ACMV were cloned as dimers into a plant transformation vector and the constructs have been integrated into tobacco protoplasts by PEG-mediated DNA transfer. In transgenic plants extrachromosomal copies of DNA1 monomers could be detected. Deletion of the coat protein-encoding gene in chimeric constructs resulted in free DNA1 copies of reduced size, and extrachromosomal recombinant molecules were detected after displacement of the coat protein-encoding region by foreign DNA fragments of comparable size. Due to the absence of the second component of ACMV, DNA2, the transgenic plants are free from viral infection symptoms which allows the establishment of healthy transformants that carry a recombinant construct in an extrachromosomal form.

Impedance Flow Cytometry: A Novel Technique in Pollen Analysis

Introduction An efficient and reliable method to estimate plant cell viability, especially of pollen, is important for plant breeding research and plant production processes. Pollen quality is determined by classical methods, like staining techniques or in vitro pollen germination, each having disadvantages with respect to reliability, analysis speed, and species dependency. Analysing single cells based on their dielectric properties by impedance flow cytometry (IFC) has developed into a common method for cellular characterisation in microbiology and medicine during the last decade. The aim of this study is to demonstrate the potential of IFC in plant cell analysis with the focus on pollen. Method Developing and mature pollen grains were analysed during their passage through a microfluidic chip to which radio frequencies of 0.5 to 12 MHz were applied. The acquired data provided information about the developmental stage, viability, and germination capacity. The biological relevance of the acquired IFC data was confirmed by classical staining methods, inactivation controls, as well as pollen germination assays. Results Different stages of developing pollen, dead, viable and germinating pollen populations could be detected and quantified by IFC. Pollen viability analysis by classical FDA staining showed a high correlation with IFC data. In parallel, pollen with active germination potential could be discriminated from the dead and the viable but non-germinating population. Conclusion The presented data demonstrate that IFC is an efficient, label-free, reliable and non-destructive technique to analyse pollen quality in a species-independent manner.

Extrachromosomal forms of CLV DNA1 in transgenic plants are inherited by symptom-free progeny

Abstract Two full length copies of Cassava latent virus (CLV) DNA1 were cloned in head to tail arrangement on a plant expression vector to evaluate the potential of CLV for the development of an extrachromosomal vector system in plants. After direct transfer of the plasmid into protoplasts of Nicotiana tabacum cv. Petit Havana SR1 extrachromosomal single-stranded (ss) and double-stranded (ds) forms of DNA1 appeared after the first cell division of protoplasts. The extrachromosomal copies could also be detected within transformants which has been regenerated from kanamycin-resistant calli. The CLV-harbouring transformants do not display any symptoms usually observed after CLV infection. Stable conservation of extrachromosomal DNA1 was observed in F1 plants derived from self-pollination and in plants regenerated from protoplasts of transformants. Our data show that dimer constructs of CLV DNA1 are attractive candidates for an extrachromosomal plant vector system.

Pepper, sweet (Capsicum annuum).

Capsicum (pepper) species are economically important crops that are recalcitrant to genetic transformation by Agrobacterium (Agrobacterium tumefaciens). A number of protocols for pepper transformation have been described but are not routinely applicable. The main bottleneck in pepper transformation is the low frequency of cells that are both susceptible for Agrobacterium infection and have the ability to regenerate. Here, we describe a protocol for the efficient regeneration of transgenic sweet pepper (C. annuum) through inducible activation of the BABY BOOM (BBM) AP2/ERF transcription factor. Using this approach, we can routinely achieve a transformation efficiency of at least 0.6 %. The main improvements in this protocol are the reproducibility in transforming different genotypes and the ability to produce fertile shoots. An added advantage of this protocol is that BBM activity can be induced subsequently in stable transgenic lines, providing a novel regeneration system for clonal propagation through somatic embryogenesis.

TET3-Mediated Demethylation in Tomato Activates Expression of a CETS Gene that Stimulates Vegetative Growth

Abstract Expression of the mammalian DNA demethylase enzyme TET3 in plants can be used to induce hypomethylation of DNA. In tomato lines that express a TET3 transgene, we observed distinct phenotypes including an increase in the length and number of leaves of primary shoots. As these changes resemble phenotypes observed in plants with strong expression of SELF PRUNING (SP), a member of the PEBP/CETS family, we investigated in TET3 lines the expression levels of members of the PEBP/CETS gene family, which affect shoot architecture and growth of sympodial units in tomato. We did not detect any changes in SP expression in TET3 lines, but for CEN1.1, a putative family member that has not been functionally characterized, we identified changes in gene expression that corresponded to hypomethylation in the upstream region. In tomato wild type, CEN1.1 is expressed in roots, petals, and shoot apices but not in mature leaves. In contrast, in TET3 transformants, the CEN1.1 gene became hypomethylated and activated in leaves. Ectopic expression of CEN1.1 in tomato caused similar phenotypes to those seen in TET3 transformants. Vegetative growth was increased, resulting both in a delay in inflorescence development and in an instability of the inflorescences, which frequently reverted to a vegetative state. Ectopic expression of CEN1.1 in Arabidopsis thaliana also caused floral repression. Our data suggest that the phenotypes observed in TET3 lines are a consequence of ectopic activation of CEN1.1, which promotes vegetative growth, and that CEN1.1 expression is sensitive to DNA methylation changes.