Zu Hauptinhalt springen
Startseite UR

Research


Fakultät für Biologie und Vorklinische Medizin
Institute of Plant Sciences
Cell Biology and Plant Biochemistry
Prof Dr. Thomas Dresselhaus - Research

General

A major characteristic of flowering plants (Angiosperms) is the double fertilization process, which was discovered more than 100 years ago by Russian and French scientists. This reproduction process takes place within the haploid female gametophyte (embryo sac; see image at the right; adapted from Diboll and Larson, 1966), which, in contrast to lower plant species, is reduced to only a few plant cells. The embryo sac consists of the two female gametes, egg (EC) and central cell (CC), two additional cells (synergids, SY) that form the egg apparatus together with the egg cell and the so called antipodal cells (AN) opposite of the egg apparatus. During fertilization, one sperm cell fuses with the egg cell generating the zygote, which initiates a coordinated developmental program of cell divisions and cell specifications, finally resulting in an embryo containing different cell types, tissues and organs. The second sperm cell fuses with the central cell, giving rise to the endosperm, known to us as the flour of cereals. 1

 

The differentiation of the female gametophyte from a single progenitor cell is, from a cell biological perspective, one of the most spectacular differentiation processes known in higher plants. Understanding the molecular mechanisms underlying the establishment of polarity and cell identity within the embryo sac, is one focus of our research team. We are also interested to identify the molecules that male (pollen) and female gametophytes use for communication and try to elucidate the mechanisms required to induce the embryonic program. We are using maize and wheat as model plant species to identify key genes involved in the processes described above. The function of some genes with homologs in the "Model Plant Species" Arabidopsis thaliana will be studied and Tripsacum dactyloides is included as a model system to investigate parthenogenesis (fertilization independent embryo development) as a component of apomixis (asexual reproduction through seeds). 2


Germline development

Gene expression and function of the female gametophyte in maize, wheat & Arabidopsis

In order to study genes expressed during female (embryo sac) gametophyte development, within the different cells of the embryo sac, as well as during the fertilization process and during early embryogenesis, we have and still are generating cDNA libraries of dissected cells of the embryo sac of maize and wheat before and shortly after fertilization. From these cDNA libraries, a few hundred differentially expressed genes have been identified (after applying diverse screening procedures), representing genes whose expression is suppressed or induced after fertilization. Some of these genes are specifically expressed in the cells of the female gametophyte. Nearly 50% of the isolated clones display significant homology to already known genes, while the remainder represent so far undescribed genes. The isolated genes are involved in many cellular processes including cell-cell communication, gene regulation, signal transduction, transcript transport, -processing and -stability, DNA replication and others.
We are now analysing the functions of a few genes in more detail: e.g. of one group which shows structural homology to defensins and might function in signaling mechanisms. Further genes encoding small peptides, which might function as signalling molecules between cells of both male and female gametophytes, are investigated using molecular, biochemical and cytological techniques. Another interesting group represents MADS box transcription factor genes functioning as repressors or activators of gene expression. Functional studies include the generation of transgenic plants, reverse genetics, mapping, biochemical (e.g. fusion proteins, 2-hybrid) and cytological studies, especially of transgenic plants. Primarily, we are interested in genes, whose products are involved in cell-cell communication, signal transduction and gene regulation.

11 1 The image on the left shows a longitudinal section of a maize kernel shortly before fertilization. The arrow marks the micropylar region of the embryo sac. The drawing shows the mature embryo sac of maize (modified after Diboll & Larson, 1966). The egg apparatus consists of the egg cell (EC) and two synergides (SY), separated from the surrounding nucellus tissue (NC) by the apical pocket (AP). The filliform apparatus (FA) consists mainly of cell wall material synthesized by the synergids. The other cells of the embryo sac are the large, highly vacuolated central cell (CC) containing two polar nuclei (PN) and a cluster of some 40 antipodal cell (AN).

Cells of the embryo sac of maize, wheat and Tripsacum are manually microdissected with glass needles from ovaries using an inverted microscope. 12

 

13 Median section through an ovule of maize. The large embryo sac cells contain numerous vacuoles. The cytoplasm of the egg cell and surrounding nucellus cells at the micropyle are RNA rich (yellow staining). This section was stained with acridine orange.

 

Dissected ovule of a transgenic maize plant expressing a ZmES4p::ZmES4-GFP fusion protein under the control of an embryo sac-specific promoter. The protein is detectable exclusively in the egg apparatus (egg cell and synergides) and weakly in the large central cell. Fusion protein accumulation is visible in the secretion region of the synergid cells adjacent to the filiform apparatus. 14


 


Fertilization mechanisms

Pollen tube growth and guidance

Once the pollen interacts with the silk/stigma and begins to hydrate, a pollen tube is extruded through the pollen tube. At a growth rate of some 0.5 cm/h in maize, the pollen tube grows rapidly through the transmitting tissue aiming to reach the female gametophyte via the micropylar region of the ovary.
We are interested in genes regulating pollen tube growth and have applied a functional genomics approach to identify oligopeptides involved in guiding the tube to the female reproductive cells. Our recent experiments have shown, that peptides secreted by the female gametophyte are involved in the final stage of pollen tube attraction.

 

21 Scanning electron micrograph of a mature pollen of maize. The germination pore is visible.

 

The transcription factor gene ZmMADS2 is expressed during pollen tube growh in maize. The image shows a germinated maize pollen hybridised with a ZmMADS2 antisense probe. 22

 

23 A peptide encoded by the ZmEA1 gene is secreted by the egg apparatus and guides the pollen tube to the micropylar region of the ovary during the final stage of tube guidance.

(a) A GFP-fusion peptide is secreted from the egg apparatus towards the extracellular space at the micropylar region.

(b) After fertilization, the GFP-fusion peptide in no longer visible.

(c) The pollen tube is guided towards the embryo sac in WT ovules.

(d) The pollen tube is not able to find its way towards the egg apparatus in transgenic maize plants, where the ZmEA1 gene is downregulated.

 


Early seed development

Early embryo and seed development

Within the frame of a genomics-project of the developing wheat grain (a cooperation with the universities of Melbourne and Adelaide), we are analysing gene activities in all cells of the wheat embryo sac before and up to three days after fertilization. Many cDNA libraries are currently established and will be analysed using bioinformatic tools.

 

Cells of the embryo sac of wheat are manually microdissected using an inverted microscope. 41

 

42 Sectioned ovary of wheat: the egg apparatus consisting of the egg cell (EC), degenerated (dSY) and viable synergide become visible. An isolated central cell containing two polar nuclei is shown on the lower right image.

 

Zygotes are isolated few hous after pollination and cultivated using a feeder system. This methods allows to get access to defined stages of zygote and early embryo development. The image shows the two-nuclei zygote stage (12 hap), the first cell division (20 hap) and a four-cxelled embryo stage (28 hap). These stages can be further cultivated to obtain more advanced embryo stages (e.g. 14 dap). 43

 

44 45

cDNA libraries will be sequenced and arrayed on glasschips to perform high throughput expression analysis. The function of few candidate genes will be studied in more detail.

 

Gene expression after fertilization
(maternal/paternal/zygotic genome activation)

We and others have shown that in maize zygotic or embryonic gene activation (ZGA, EGA) occurs shortly after fertilization. This is in contrast to investigations in Arabidopsis, where EGA occurs not within the first three days after fertilization, although this is still a controverse matter of debate. In most animal species, EGA occurs when the embryo consists already of few (mammals) and up to thousands of cells (e.g. frog and fruit fly). In animals, it was found that many genes are silenced (imprinted) either on the male or female genome, respectively, and thus only one of the homologs is active during early embryo development. Imprinting also occurs in plants and a number of mainly maternal imprinted genes have been identified recently in maize and Arabidopsis. Plant imprinted genes seem to be mainly involved in endosperm development.
We are performing reciprocal crossings of inbred and transgenic
lines of maize to study maternal and paternal gene expression shortly after fertilization. In addition, parthenogenetic lines (no paternal contribution to the embryonic genome) of Tripsacum dactyloides, a close relative of maize, are used to study differences in gene expression pattern between sexual and parthenogenetic embryo development. We are using a combination of microdissection techniques (described elsewhere) with molecular methods (e.g. SNP analysis: single nucleotide polymorphisms) to distinguish between homologous paternal genes.

 

31 The paternal genome of maize is activated shortly after fertilization. Two pollen tubes of a transgenic maize line shortly before and after penetrance of the degenerating synergide of a non-transgenic line: the two sperm cells of one pollen tube have fertilized egg and central cell, respectively, visible due to paternal marker gene expression in the egg apparatus as well as in the large central cell.


Understanding parthenogenesis as a component of apomixis

Understanding parthenogenesis as a component of apomixis

Apomixis describes asexual types of reproduction through seeds and is wide spread in nature, naturally occurring in hundreds of plant species, especially in the Asteraceae (e.g. Common Dandelion), Rosaceae and the grasses. Apomictic seeds contain embryos, which are genetic copies of the mother plant. This is particularly interesting if the mother plant is a hybrid itself. There is a great commercial interest to use apomixis in plant breeding and seed production, because it would allow easy marketing of heterozygous hybrid plant material and an enorming decrease of breeding times and thus the possibility to generate crop plants adapted to numerous climatic and environmental conditions..
Our research activities in the field of apomixis focus on the comparison of gene expression pattern in sexual and parthenogenetic eggs with the aim to identify key genes involved in the induction of the embryonic program and/or to discover genes maintaining the undifferentiated stem cell character of the sexual egg cell. We are using maize and Tripsacum dactyloides (different sexual and apomictic lines) as model species.

Apomixis for hybrid seed production.

Parental inbred lines (P1 and P2) are crossed to generate hybrid seeds. Due to the heterosis-effect, F1 plants are more vigorous than parent plants and produce a higher yield. Genetic segregation occurs in F2 plants - but not in apomictic plants.

 

51

 

 

52 The mode of reproduction can be determined applying the flow cytometric seed screen (FCSS) using extracts of single mature seeds. The hight and relative position of peaks originating from seed nuclei in extracts after staining determine the reproduction mode: the example shows a seeds of a hexaploid Tripsacum dactyloides-linie, where the embryo developed parthenogenetically and the endosperm after fertilization of the central cell (pseudogamy).

 

Microdissection of reproductive cells from Tripsacum dactyloides for molecular analyses.

(a & b) male and female flowers, (c & d) ovule, (e) parthenogenetic egg cell, (f) synergids and (g) parthenogenetic embryo.

53


Flower-pathogen interactions

Page under construction.


Heat stress and reproduction

Page under construction.


Evolution of reproductive mechanisms

Page under construction.


Regulation of cell polarity and identity

Page under construction.


mRNP formation and translational control

Page under construction.


Cellular cross-talk and signaling

Page under construction.

  1. Fakultät für Biologie und Vorklinische Medizin
  2. Faculty Research

Prof. Dr. Thomas Dresselhaus

Cell Biology and Plant Biochemistry
 
University of Regensburg

Lab1

Universitätsstraße 31
93053 Regensburg
Germany

Phone: +49 (0)941 943-3016
Fax: +49 (0)941 943-3352
E-Mail