In vitro fertilization with isolated higher plant gametes.

Methods have been developed to isolate gametes of higher plants and to fertilize them in vitro. Zygotes, embryos, fertile plants and endosperm can now be obtained from in vitro fusion of pairs of sperm and egg cells and of pairs of sperm and central cells, respectively. This allows examination of the earliest developmental processes precisely timed after fertilization.

The auxin-induced K(+) channel gene Zmk1 in maize functions in coleoptile growth and is required for embryo development.

The transcript level and in turn protein density of the K(+)-uptake channel ZMK1 in maize (Zea mays) coleoptiles is controlled by the phytohormone auxin. ZMK1 is involved in auxin-regulated coleoptile elongation as well as gravi- and phototropism. To provide unequivocal evidence for the role of ZMK1 in these elementary processes we screened for maize plants containing a Mutator-tagged Zmk1 gene.

Epigenetic asymmetry of imprinted genes in plant gametes.

Plant imprinted genes show parent-of-origin expression in seed endosperm, but little is known about the nature of parental imprints in gametes before fertilization. We show here that single differentially methylated regions (DMRs) correlate with allele-specific expression of two maternally expressed genes in the seed and that one DMR is differentially methylated between gametes. Thus, plants seem to have developed similar strategies as mammals to epigenetically mark imprinted genes.

Construction and screening of subtracted cDNA libraries from limited populations of plant cells: a comparative analysis of gene expression between maize egg cells and central cells.

The analysis of cell type-specific gene expression is an essential step in understanding certain biological processes during plant development, such as differentiation. Although methods for isolating specific cell types have been established, the application of cDNA subtraction to small populations of isolated cell types for direct identification of specific or differentially expressed transcripts has not yet been reported. As a first step in the identification of genes expressed differentially between maize egg cells and central cells, we have manually isolated these types of cell, and applied a suppression-subtractive hybridization (SSH) strategy.

Phytosulphokine gene regulation during maize (Zea mays L.) reproduction.

The sulphated pentapeptide phytosulphokine (PSK) was identified as a substance that promotes cell division in low-density suspension cultures and has been implicated in various aspects of tissue differentiation in plants. The peptide is derived from PSK precursor proteins that are encoded by small gene families. The physiological roles of PSK are still not clearly defined and little is known about expression of members of the PSK precursor gene family in any plant species.

Identification of genes that are up- or down-regulated in the apical or basal cell of maize two-celled embryos and monitoring their expression during zygote development by a cell manipulation- and PCR-based approach.

In higher plants, a zygote generally divides asymmetrically into a two-celled embryo. As in planta, maize zygotes produced by in vitro fertilization of an egg cell with a sperm cell also develop into an asymmetrical two-celled embryo that consists of a small plasma-rich apical cell and a large vacuolized basal cell. Subsequently, via zygotic embryogenesis, a proembryo and a transition phase embryo are formed from the two-celled embryo.

Identification of major proteins in maize egg cells.

In most flowering plants, the female gametophyte develops in an ovule deeply embedded in the ovary. Through double fertilization, the egg cell fuses with the sperm cell, resulting in a zygote, which develops into the embryo. In the present study, we analyzed egg cell lysates by polyacrylamide gel electrophoresis and subsequent mass spectrometry-based proteomics technology, and identified major protein components expressed in the egg cell.

Extensive maternal DNA hypomethylation in the endosperm of Zea mays.

A PCR-based genomic scan has been undertaken to estimate the extent and ratio of maternally versus paternally methylated DNA regions in endosperm, embryo, and leaf of Zea mays (maize). Analysis of several inbred lines and their reciprocal crosses identified a large number of conserved, differentially methylated DNA regions (DMRs) that were specific to the endosperm. DMRs were hypomethylated at specific methylation-sensitive restriction sites upon maternal transmission, whereas upon paternal transmission, the methylation levels were similar to those observed in embryo and leaf.

Isolation of AtSUC2 promoter-GFP-marked companion cells for patch-clamp studies and expression profiling.

K+ channels control K+ homeostasis and the membrane potential in the sieve element/companion cell complexes. K+ channels from Arabidopsis phloem cells expressing green fluorescent protein (GFP) under the control of the AtSUC2 promoter were analysed using the patch-clamp technique and quantitative RT-PCR. Single green fluorescent protoplasts were selected after being isolated enzymatically from vascular strands of rosette leaves.

Paternal mRNA and protein synthesis coincides with male chromatin decondensation in maize zygotes.

Decondensation of the male genome after fertilization is a prerequisite for replication and transcription. Cytological analysis has revealed decondensation of the male chromatin to commence immediately after karyogamy and progress rapidly, pointing to an early start of transcription. To investigate early transcription from the paternal genome in maize zygotes, we generated transgenic plants containing green fluorescent protein (GFP) under control of the 35S promoter.