1 Principal Research Accomplishments:
1.1 Characterization of viroid-protein complexes
During my Diploma work I have used biochemical and biophysical methods to characterize viroid-protein complexes. In cooperation with my advisors, Prof. Dr. Detlev Riesner and Dr. Noemi Lukacs, I could show that specific viroid-protein complexes can be reconstituted in vitro (Klaff et al., 1989).
1.2 Development of methods for molecular biology
During all early stages of my scientific career I have developed new methods for molecular biology. During my Diploma work I have established a procedure for the isolation of proteins that bind to viroids, which is based on affinity chromatography. Moreover, I have developed a gel retardation assay for the determination of the degree of biotinylation of nucleic acids (Theißen et al., 1989). During my Ph.D. work I have developed a quantitative assay for transcriptional pausing of DNA-dependent RNA polymerases in vitro. The assay was used to measure pause strengths on supercoiled templates in the leader region of the E. coli rrnB operon in the presence and the absence of the transcription factor NusA (Theißen et al., 1990c). Moreover, I have shown that temperature-gradient gel electrophoresis can be used for the detection and characterization of curved DNA-fragments (Theißen et al., 1993a). In cooperation with a Ph.D. and a diploma student I have developed a linker PCR procedure for preparing repetitive DNA-free probes from genomic clones, which is especially efficient for members of gene families. Employing this procedure, chromosomal map positions of MADS-box genes could be determined quite efficiently in recombinant inbred lines of maize (Zea mays ssp. mays) (Fischer et al., 1995a). Moreover, we developed a protocol for efficient expression analysis of multigene families, called RC4D (Fischer et al., 1995b; Fischer et al., 1997; Theißen and Fischer, 1997). The method combines RFLP technology with a gene family-specific version of mRNA differential display. RC4D enables a detailed, high-resolution expression analysis of known gene family members as well as the identification and characterization of new ones. RC4D was used to identify MADS-box genes that are differentially expressed between the male and female inflorescences of maize, and between the female inflorescences of maize and teosinte. More recently, a simple method for predicting the functional differentiation of duplicate genes has been developed and applied to MIKC-type MADS-domain proteins (Nam et al., 2005).
1.3 Studies on the biogenesis of ribosomes in E. coli
The most significant accomplishment during my Ph.D. work was demonstrating that some sequence elements in the leader region of a ribosomal RNA operon in E. coli assist the structure formation of functional 30S ribosomal subunits, although they are not part of the mature ribosome (Theißen et al., 1990a,b; Theißen et al., 1993b). The ribosomal RNA leader thus works as a molecular chaperone, although it is not a protein, but an RNA (Theißen et al., 1993b). Although that was quite a surprising finding at its time, similar mechanisms have meanwhile also been demonstrated to play a role in ribosome formation in eukaryotes.
1.4 MADS-box genes in green plants: establishing a new research paradigm for evo-devo
The role of HOX genes in animal evolution has found wide recognition. But how general are the findings in this system for the evolution outside of animals? Since 1992 my group has worked on establishing MADS-box gene phylogeny and plant development as a new research paradigm for evolutionary developmental biology (‘evo-devo’). It is a corollary of evo-devo that the same genes which control the development of organs played also important roles during the evolution of these organs (Theißen and Saedler, 1995). Homeotic organ identity genes belonging to the MADS-box gene family sculpt the structure of dicotyledonous flowers. The establishment of floral homeotic genes and changes in their regulation or function, therefore, may have contributed to the establishment and structural evolution of flowers. Understanding the phylogeny of MADS-box genes might thus help us to gain a better understanding of flower origin and diversity (Theissen et al., 2000; Theien, 2001).
Phylogeny reconstructions revealed that the MADS-box gene family is composed of several defined gene clades (for contributions from my group, see e.g. Theißen et al., 1996; Münster et al., 1997; Theissen et al., 2000; Becker and Theißen, 2003). Most clade members share highly related functions and similar expression patterns. For example, the MADS-box genes providing the floral homeotic functions A, B and C each fall into separate clades, namely SQUAMOSA- (A), DEFICIENS- or GLOBOSA- (B), and AGAMOUS-like genes (C). The genes determining ovule identity (termed D-function genes) also belong to the clade of AGAMOUS-like genes, and ‘E-function genes’ (Theißen, 2001) are members of the clade of AGL2-like (or SEPALLATA-like) genes. Therefore, the establishment of the above mentioned gene clades was probably an important event towards the establishment of the floral homeotic functions. Thus questions arise such as: when did these gene clades originate during evolution? How and when did some of their members become floral organ identity genes? How did the phylogeny of these genes influence the evolution of the flower?
To answer these questions we started to characterize the MADS-box gene family in phylogenetic informative taxa. In addition to our interest in the monocotyledonous plant maize (see below), we also studied MADS-box genes in the monocots lily and tulip (for more details, see Current Research) (Theissen et al., 2000; Kanno et al., 2003).
In addition, we analyzed MADS-box genes in some gymnosperms, especially Gnetum gnemon. We have isolated a large number of diverse MADS-box genes from Gnetum, among them putative orthologs of floral homeotic B- and C-function genes, and of a hitherto unknown sister clade of the B-function genes (Winter et al., 1999; Becker et al., 2000, 2002). These genes helped us to develop detailed models about the origin of the floral homeotic functions, the floral organs, and the flower as a compound structure (see e.g. Theißen et al., 2002; Theißen and Becker, 2004b). Studies on the function of these genes in vitro, in vivo and in planta, employing e.g. gel retardation assays, the yeast two-hybrid system and Arabidopsis transformation, indicated conservation as well as differences in gene expression, function and protein-protein interaction patterns (Winter et al., 2002a,b; Becker et al., 2003). A key aspect of our model of flower origin may be tested by large scale comparisons of gene expression patterns in flowers and male as well as female reproductive cones of gymnosperms. This approach will be strongly facilitated by applying some of the novel approaches of genomics, such as gene expression microarray studies (Soltis et al., 2002). We cooperated with groups in Australia and Sweden to isolate the first class B gene orthologs from conifers, i.e. Pinus radiata and Picea abies (Mouradov et al., 1999; Sundström et al., 1999). Using some MADS-box genes from Gnetum and their orthologs from conifers and angiosperms as molecular markers we have obtained evidence that the gnetophytes are more closely related to conifers than to angiosperms, which is in contradiction to textbook interpretations of morphological traits for almost a century (Winter et al., 1999).
To gain insights into the function of MADS-box genes in non-seed plants we have isolated members of that gene family from the ferns Ceratopteris and Ophioglossum, and the moss Physcomitrella patens. A characterization of the MADS-box gene family in the ferns Ceratopteris and Ophioglossum suggested that the most recent common ancestor of ferns and seed plants about 400 million years ago contained at least two different MADS-box genes which were homologs, but not orthologs of floral homeotic genes (Münster et al., 1997, 2002). These genes probably had expression patterns and functions that were more general than those of the highly specialized floral homeotic genes from extant flowering plants. In the moss Physcomitrella patens we identified a couple of ‘classical’ MIKC-type genes, but also members of a novel class (termed MIKC*) (Henschel et al., 2002). Work to knock-out these genes via homologous recombination is currently underway in the group of my former postdoctoral co-worker Thomas Münster at the MPIZ in Cologne (Germany). Our work on non-seed plant MADS-box genes also helped to understand the deep origin of MIKC-type genes in green (charophyte) algae (Tanabe et al., 2005).
1.5 MADS-box genes as tools for crop plant design
Most human food is derived from flower parts or products, such as fruits and grains. Understanding the genetic basis of flower development, therefore, may help us to transform inflorescences and flowers according to our desires. The time to flowering, and the number and structure of the inflorescences and flowers are critical parameters that determine, at least in part, where a crop plant can be grown and how many fruits or grains it may produce. In principle, it should be possible to create plants that flower earlier or later than wild-type by changing the expression of floral meristem identity genes. In addition, plants that produce more fruits or grains might be established by changing the expression of floral organ identity genes or other developmental control genes that influence the architecture of flowers or inflorescences. MADS-box genes contribute significantly to the development of inflorescences and flowers, e.g. by acting as floral meristem or organ identity genes. Thus in contrast to "convential" plant genetic engineering, which tries to optimize traits such as disease resistance or food quality, with MADS-box genes to hand it might be possible to "design" crop plants to have radically altered structural properties.
A first essential step towards developing MADS-box genes as tools for such a "radical crop design" is to characterize the members of this gene family in suitable crop plants. A few years ago, a critical question was whether the MADS-box genes of monocots (comprising the globally most important crop plants, such as wheat, rice and maize) are structurally similar and functional equivalent to those of the dicotyledonous model plants. Meanwhile, we and several other laboratories have demonstrated that monocotyledonous plants such as maize and rice have indeed MADS-box genes that are orthologous to those of core eudicots (for contributions from my group see e.g. Theißen et al. 1995, 1996; Fischer et al. 1995a,b; Cacharrón et al. 1999; Münster et al., 2001, 2002; European Patent Application No. 98 12 4335.5: "Novel MADS-box genes and uses thereof"; International patent Application PCT/EP99/10116).
We have learned from our studies that maize offers a wealth of MADS-box genes with diverse expression patterns that are highly specific for different morphological structures such as floral organs or meristems, or complete flower or spikelet primordia (Münster et al., 2002). Based on what is known from eudicotyledonous MADS-box genes, a similar diversity of functions, e.g. in the developmental specification of different floral structures, can be expected. A transgenic change in the expression of maize MADS-box genes, therefore, might indeed generate drastically changed plant structures useful for a future generation of agriculture. Moreover, the defined spatio-temporal expression patterns of the maize MADS-box genes promise that the promoters of these genes could be used to accurately direct the expression of arbitrary transgenes into a diversity of plant structures.
To fully develop the agronomic potential of the MADS-box gene family of maize we worked on a comprehensive isolation and characterization of its members. In a project that, in its initial phase, was supported by the German Ministry of Science and four industrial companies we have cloned and sequenced eventually more than 30 different MADS-box genes from maize (Münster et al., 2001, 2002). The expression patterns of the genes were determined by Northern and in situ hybridizations. The map positions of the genes were determined in order to find out whether they coincide with known mutant loci. Information about the functions of some genes was obtained by the analysis of such mutants, transgenic studies and reverse genetics. The data that we have accumulated helped us to find those genes that are most suitable for a "radical crop design" by transgenic technology, as defined above. With ZMM8, for example, a MADS-box gene was identified which is involved in spikelet development. A reduction of ZMM8 gene function in transgenic maize plants led to an increase in the number of florets per spikelet. In rice and some other cereals, where normally only one floret per spikelet develops, this principle might be applied to increase crop yield (European Patent Application No. 98 12 4335.5: "Novel MADS-box genes and uses thereof"; International patent Application PCT/EP99/10116).
In addition to findings which might facilitate a future crop plant design our studies on MADS-box genes in maize yielded quite a number of interesting results by serendipity. For example, we could demonstrate that the phenotype of one of the most widely known maize mutants, Tunicate1 (or pod corn), is due to the ectopic expression of a “vegetative” MADS-box gene (ZMM19) in the inflorescences of maize. We could define changes in the promoter region of the Tunicate1 gene as the molecular cause of the changes in its expression pattern (manuscript in preparation).
2 Current Research
My group is interested in understanding the mechanisms that generate evolutionary novelties. Towards that goal we investigate as to how the phylogeny of developmental control genes encoding transcription factors contributed to the evolution of complex morphological structures. Our major model system is the phylogeny of MADS-box genes and their role in the evolution of land plants. Furthermore, we have an interest in the role of homeotic mutants in evolution, and, more generally, in finding out as to whether saltational events play a role in evolution. For further information about the projects currently carried out in my lab, please check our "research" page.
3 Some Plans for the Future
Ongoing projects (see our research page) will remain important research activities also in the near and intermediate future. In addition, we are currently initiating the following research projects:
3.1 Towards an evolutionary developmental genetics of gymnosperms: a case study involving the Acrocona mutant of Norway spruce
Understanding the origin of the angiosperm flower requires that we better understand the developmental genetics of extant gymnosperms. Acrocona is a homeotic and heterochronic mutant of Norway spruce (Picea abies) in which female cones develop dramatically earlier than in the wild-type. Cloning and molecular developmental characterization of the Acrocona locus of Norway spruce will provide important insights into the reproductive developmental genetics of conifers. Work on the topic is facilitated by a segregating population of Acrocona that our cooperation partner, Matthias Fladung (Großhansdorf), developed more than 10 years ago.
3.2 Fast evolution of functionally relevant sequence repeats in MADS-domain transcription factors in Brassicaceae and Solanaceae
Some floral homeotic proteins contain simple sequence repeats such as PolyQ stretches that are quite polymorphic. We want to test as to whether the dynamic evolution of such stretches facilitates the morphological evolution of flowering plants, with a focus on Brassicaceae and Solanaceae.
3.3 MADS-box genes as tools for crop plant design in maize and rice
This project will follow up some of the most promising projects of point 1.5 (see above) on maize; analogous research is planned for rice (cooperations with groups in China have started).
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