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.

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 isolated egg and central cell, fertilized and cultured in vitro, are able to self-organize apart from each other and without mother tissue in the typically manner. Thus, this system is a powerful and unique model for studies of early zygotic embryogenesis and endosperm development. The underlying processes are now comparatively studied in detail by investigations of expression of genes and their corresponding proteins. The use of these techniques opens new avenues in fundamental and applied research in the areas of developmental and reproductive plant biology.

WOX gene phylogeny in Poaceae: a comparative approach addressing leaf and embryo development.

The phylogeny based on the homeodomain (HD) amino acid sequence of the WOX (WUSCHEL-related homeobox gene family) was established in the 3 major radiations of the Poaceae family: Pooideae (Brachypodium distachyon), Bambusoideae (Oryza sativa), and Panicoideae (Zea mays). The genomes of all 3 grasses contain an ancient duplication in the WOX3 branch, and the cellular expression patterns in maize and rice indicate subfunctionalization of paralogues during leaf development, which may relate to the architecture of the grass leaf and the encircling of the stem. The use of maize WOX gene family members as molecular markers in maize embryo development for the first time allowed us to visualize cellular decisions in the maize proembryo, including specification of the shoot/root axis at an oblique angle to the apical-basal polarity of the zygote. All molecular marker data are compatible with the conclusion that the embryonic shoot/root axis comprises a discrete domain from early proembryo stages onward. Novel cell fates of the shoot and the root are acquired within this distinct morphogenic axis domain, which elongates and thus separates the shoot apical meristem and root apical meristem (RAM) anlagen in the maize embryo.

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. In a site-selected approach, we were able to identify three independent alleles of Mutator-transposon insertions in Zmk1. zmk1-m1::Mu1 plants were characterised by a Mu1 transposon inside intron 1 of ZMK1. When we analysed the Zmk1-transcript abundance in growing coleoptiles of these homozygous mutants, however, we found the K(+)-channel allele overexpressed. In consequence, elevated levels of K(+)-channel transcripts resulted in a growth phenotype as expected from more efficient K(+)-uptake, representing a central factor for turgor formation. Following Zmk1 expression during maize embryogenesis, we found this K(+)-channel gene constitutively expressed throughout embryo development and upregulated in late stages. In line with a vital role in embryogenesis, the mutations of exon 2 and intron 2 of Zmk1-zmk1-m2::Mu8 and zmk1-m3::MuA2-caused a lethal, defective-kernel phenotype. Thus, these results demonstrate the central role of the auxin-regulated K(+)-channel gene Zmk1 in coleoptile growth and embryo development.

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. In the present study, we focused on identifying genes that were up- or down-regulated only in the apical or basal cell of two-celled maize embryos after fertilization. First, a procedure for isolating the apical and basal cells from two-celled embryos was established, and subsequently cDNAs were synthesized from apical cells, basal cells, egg cells, two-celled embryos and multicellular embryos. These cDNAs were used as templates for polymerase chain reaction (PCR) with randomly amplified polymorphic DNA (RAPD) primers. Genes with specific expression patterns were identified, and these expression patterns were categorized into six groups: (1) up-regulated only in the apical cell after gamete fusion; (2) up-regulated only in the basal cell after gamete fusion; (3) up-regulated in both the apical and basal cells after gamete fusion; (4) down-regulated only in the apical cell after gamete fusion; (5) down-regulated only in the basal cell after gamete fusion; and (6) constitutively expressed in the egg cell and embryos. In addition, it was revealed that the genes up-regulated in the apical or basal cell (genes in groups 1 and 2) were already expressed in the early zygote, providing the possibility that the transcripts from these genes are localized to the putative apical or basal region of the zygote, or that the transcripts are rapidly degraded in one of the daughter cells after zygotic cell division.

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. In this study, highly regulated tissue and cell type-specific expression are described for four PSK genes from maize (Zea mays L.) in female and male gametophytes, and during seed development. ZmPSK1 and ZmPSK3 were specifically and differentially expressed in cells of female and male gametophytes and in female and male gametes. In anthers ZmPSK1 or ZmPSK3 transcripts were found, for example, at high levels in secretory tapetal cells which support developing microspores. ZmPSK1 mRNA was abundant in mature pollen including sperm cells. ZmPSK1 and ZmPSK3 transcripts were also detected in egg and central cells of the female gametophyte and ZmPSK1 mRNA was present in synergids, indicating that the PSK peptide probably plays a role during gametogenesis, pollen germination, and fertilization. In developing maize kernels all four ZmPSK genes were expressed, albeit with striking differences in their expression patterns. It is proposed here that PSK is required for numerous but defined processes during gametophyte and early sporophyte development. In general, PSK availability appears to be controlled through transcriptional regulation in a tissue and cell type-specific and development-dependent manner.

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. After microarray screening of 1030 cDNAs obtained from the subtracted libraries, we identified 340 differentially expressed clones. Of these, 142 were sequenced, which resulted in the identification of 62 individual cDNAs. The expression patterns of 20 cDNAs were validated by quantitative RT-PCR, through which we identified five transcripts with cell type-specific expression. The specific localization of some of these transcripts was also confirmed by in situ hybridization on embryo sac sections. Taken together, our data demonstrate the effectiveness of our approach in identifying differentially expressed and cell type-specific transcripts of relatively low abundance. This was also confirmed by the identification of previously reported egg cell- and central cell-specific genes in our screen. Importantly, from our analysis we identified a significant number of novel sequences not present in other embryo sac or, indeed, in other plant expressed sequence tag (EST) databases. Thus, in combination with standard EST sequencing and microarray hybridization strategies, our approach of differentially screening subtracted cDNAs will add substantially to the expression information in spatially highly resolved transcriptome analyses.

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. Single transgenic sperm cells from these plants were used to fertilize isolated wild-type egg cells in vitro. These sperm cells did not contain gfp transcripts. Appearance of gfp mRNA, 4 h after fertilization, was coincident with decondensation of the male chromatin, and clearly demonstrates early accessibility to the transcriptional machinery of at least a part of the male genome. Translational activity in early zygotes was evident 6 h after fertilization, as demonstrated by measurable levels of GFP fluorescence signal. Using a similar strategy, we also demonstrated activity of the paternal genome early in endosperm development. These findings may exclude any global mechanism of silencing the entire paternal genome over this period, and make an almost immediate paternal contribution to zygote and early endosperm development conceivable. These data are also considered in the perspective of current views of genome activation in the zygotes and young embryos of animals.

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. The identified proteins included three cytosolic enzymes of the glycolytic pathway, glyceraldehyde-3-phosphate dehydrogenase, 3-phosphoglycerate kinase and triosephosphate isomerase, two mitochondrial proteins, the ATP synthase beta-subunit and an adenine nucleotide transporter, and annexin p35. In addition, expression levels of these proteins in the egg cell were compared with those in the early embryo, the central cell and the suspension cell. Annexin p35 was highly expressed only in the egg cell, and glyceraldehyde-3-phosphate dehydrogenase, 3-phosphoglycerate kinase and the adenine nucleotide transporter were expressed at higher levels in egg cells than in central and cultured cells. These results indicate that annexin p35 in the egg cell and zygote is involved in the exocytosis of cell wall materials, which is induced by a fertilization-triggered increase in cytosolic Ca2+ levels, and that the egg cell is rich in an enzyme subset for the energy metabolism.

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. Maternal hypomethylation was extensive and offers a likely explanation for the 13% reduction in methyl-cytosine content of the endosperm compared with leaf tissue. DMRs showed identity to expressed genic regions, were observed early after fertilization, and maintained at a later stage of endosperm development. The implications of extensive maternal hypomethylation with respect to endosperm development and epigenetic reprogramming will be discussed.

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. Companion cell protoplasts, which could be recognized by their nucleus, vacuole and chloroplasts, and by their expression of the phloem-specific marker genes SUC2 and AHA3, formed the basis for a cell-specific cDNA library and expressed sequence tag (EST) collection. Although we used primers for all members of the Shaker K+ channel family, we identified only AKT2, KAT1 and KCO6 transcripts. In addition, we also detected transcripts for AtPP2CA, a protein phosphatase, that interacts with AKT2/3. In line with the presence of the K+ channel transcripts, patch-clamp experiments identified distinct K+ channel types. Time-dependent inward rectifying K+ currents were activated upon hyperpolarization and were characterized by a pronounced Ca2+-sensitivity and inhibition by protons. Whole-cell inward currents were carried by single K+-selective channels with a unitary conductance of approximately 4 pS. Outward rectifying K+ channels (approximately 19 pS), with sigmoidal activation kinetics, were elicited upon depolarization. These two dominant phloem K+ channel types provide a versatile mechanism to mediate K+ fluxes required for phloem action and potassium cycling.