Na+ transport by the neural glutamine transporter ATA1.

Transfer of glutamine between astrocytes and neurons is an essential part of the glutamate-glutamine cycle in the brain. Here we have investigated how the neural glutamine transporter (rATA1/GlnT) works. Rat ATA1 was expressed in Xenopus laevis oocytes and examined using two-electrode voltage-clamp recordings, ion-sensitive microelectrodes and tracer flux experiments. Glutamine transport via rATA1 was electrogenic and caused inward currents that did not reverse at positive holding potentials. Currents were induced by a variety of neutral amino acids in the following relative order Ala>Ser/Gln/Asn/His/Cys/Met >MeAIB/Gly>Thr/Pro/Tyr/Val, where MeAIB is the amino acid analogue N-methylaminoisobutyric acid. The uptake of glutamine and the corresponding currents depended on Na+ and pH. Hill-coefficient and flux studies with 22NaCl indicated a cotransport stoichiometry 1 Na+ per transport cycle. The transporter also showed uncoupled Na+ transport, particularly when alanine was used as the substrate. Although substrate uptake increased strongly with increasing pH, no change of intracellular pH was observed during transport. A decrease of the intracellular pH similarly inhibited glutamine transport via ATA1, suggesting that the pH dependence was an allosteric effect on the transporter.

The maize MADS box gene ZmMADS3 affects node number and spikelet development and is co-expressed with ZmMADS1 during flower development, in egg cells, and early embryogenesis.

MADS box genes represent a large gene family of transcription factors with essential functions during flower development and organ differentiation processes in plants. Addressing the question of whether MADS box genes are involved in the regulation of the fertilization process and early embryo development, we have isolated two novel MADS box cDNAs, ZmMADS1 and ZmMADS3, from cDNA libraries of maize (Zea mays) pollen and egg cells, respectively. The latter gene is allelic to ZAP1. Transcripts of both genes are detectable in egg cells and in in vivo zygotes of maize. ZmMADS1 is additionally expressed in synergids and in central and antipodal cells. During early somatic embryogenesis, ZmMADS1 expression is restricted to cells with the capacity to form somatic embryos, and to globular embryos at later stages. ZmMADS3 is detectable only by more sensitive reverse transcriptase-PCR analyses, but is likewise expressed in embryogenic cultures. Both genes are not expressed in nonembryogenic suspension cultures and in isolated immature and mature zygotic embryos. During flower development, ZmMADS1 and ZmMADS3 are co-expressed in all ear spikelet organ primordia at intermediate stages. Among vegetative tissues, ZmMADS3 is expressed in stem nodes and displays a gradient with highest expression in the uppermost node. Transgenic maize plants ectopically expressing ZmMADS3 are reduced in height due to a reduced number of nodes. Reduction of seed set and male sterility were observed in the plants. The latter was due to absence of anthers. Putative functions of the genes during reproductive and vegetative developmental processes are discussed.

ZmES genes encode peptides with structural homology to defensins and are specifically expressed in the female gametophyte of maize.

All four members of a gene family, which are highly expressed in the cells of the female gametophyte (ZmES1--4: Zea mays embryo sac), were isolated from a cDNA library of maize egg cells. High expression of ZmES genes in the synergids around the micropylar region was detected in thin sections of maize ovaries. Single-cell RT--PCR analyses with the various cells of the female gametophyte confirmed the expression in synergids and also showed expression in the egg cell and central cell, and low expression in the antipodals. The expression of the whole gene family is suppressed after fertilization of the embryo sac, and expression in two-cell or later embryo stages or other tissues of maize could not be detected. In order to investigate ZmES mRNA gradients in the highly polarized and vacuolized cells of the maize embryo sac, a whole-mount in situ protocol with isolated single cells was developed: as for total RNA, ZmES transcripts are uniformly distributed in the cytoplasm of egg cell, synergids and central cell. ZmES genes encode small, cysteine-rich proteins with an N-terminal signal peptide, probably for translocation into the embryo sac cell wall. The four ZmES proteins display high sequence identity with each other, and the proposed tertiary structure of the mature peptides is similar to that of plant and animal defensins. The function of ZmES1-4 during the fertilization process is discussed.

Novel ribosomal genes from maize are differentially expressed in the zygotic and somatic cell cycles.

We have isolated cDNAs representing more than 50 different genes from libraries of unfertilised egg cells and zygotes of maize, expression of which is up- or downregulated after in vitro fertilisation (IVF). Among the cDNAs isolated are seven which encode proteins that are probably involved in translation and two encoding proteins probably involved in DNA replication. The latter genes are strongly induced on fertilisation. This indicates that zygotic gene activation (ZGA) - the switch from maternal to embryonic control of development - occurs in the zygote shortly after fertilisation in higher plants - earlier than in animal systems so far investigated. Four novel transcripts for ribosomal proteins (S21A, S21B, L39, P0) involved in ribosome biosynthesis and translation were analysed in more detail. The expression of all four genes correlates with cell division activity and is strongly induced during the G1 phase of the somatic cell cycle. A different mode of regulation operates in the first embryonic cell cycle: relatively large amounts of transcript are stored in the unfertilised egg cells, and by 18 h after IVF, two ribosomal genes are induced while a third is downregulated. These results indicate that using the combination of single-cell culture techniques with novel molecular methods, it is possible to isolate and study numerous genes expressed in female gametes and zygotes of higher plants. The detailed analysis of the four ribosomal protein genes demonstrates that the zygotic and somatic cell cycles are differentially regulated.

Endosperm development after fusion of isolated, single maize sperm and central cells in vitro

We demonstrate here the possibility of endosperm development in vitro after the fusion of pairs of an isolated sperm and an isolated central cell of maize. The occurrence of karyogamy and the time course of the fusion of sperm and central cell nuclei are presented. The fusion of the sperm nucleus occurred either with one of the two polar nuclei or with the secondary nucleus and was completed within 2 hr after in vitro cell fusion. The in vitro study of early events after cell and nuclear fusion indicates that the resulting primary endosperm cell develops into a characteristic tissue capable of self-organization apart from the mother tissue. The technology presented here opens the way for new cellular and molecular studies, especially of early events after sperm and central cell fusion. These studies should lead to a better understanding of the processes of double fertilization and endosperm development.

Isolation of viable egg cells of rape (Brassica napus L.).

The isolation of viable egg cells of rape (Brassica napus L.) has been achieved from microdissected ovules. The non-gametic cells of the embryo sac, synergids and central cells have also been isolated. Their morphology corresponded to that of these cells in situ, making a discrimination from isolated sporophytic cells possible. Two hours after isolation the egg cells were still viable. Viable egg cells have been reproducibly isolated with a frequency of 25% per dissected ovule.

Isolation of a full-length cDNA encoding calreticulin from a PCR library of in vitro zygotes of maize.

A full-size cDNA clone (1614 bp) encoding calreticulin was isolated from a PCR-based cDNA library of maize in vitro zygotes. Calreticulin is a major Ca2+ storage protein located mainly in the lumen of the endoplasmic reticulum but also in the nucleus and/or cytoplasm of some cells. A differential screening between cDNA libraries originating from 104 in vitro zygotes (18 h after in vitro fertilization) and 128 unfertilized egg cells was performed to isolate newly expressed genes or genes expressed more abundantly after fertilization. The expression of the isolated cDNA clone is enhanced after fertilization and strongly correlated to cell division. Sequence comparison to a shorter maize calreticulin cDNA isolated from a PCR based cDNA library construction from a few plant cells [12]. It is further shown that calreticulins in maize are probably transcribed from a small gene family differentially expressed in abundance in diverse tissues. The deduced amino acid sequence encodes an acidic protein (pI 4.17) of 48 kDa sharing 77-92% and 50-54% homology to other plant and animal calreticulins, respectively. The described calreticulin gene represents to our knowledge the first cDNA clone isolated from a RT/PCR cDNA library originating from only a few plant cells and is the first gene isolated from zygotes of higher plants.

Characterisation of isolated egg cells, in vitro fusion products and zygotes of Zea mays L. using the technique of image analysis and confocal laser scanning microscopy.

Changes in membrane Ca2+, calcium receptor protein calmodulin, endoplasmic reticulum (ER), mitochondria and cellulose in unfixed, living, isolated egg cells and fusion products of pairs of one egg and one sperm cell of Zea mays L. have been investigated using chlorotetracycline, fluphenazine, immunocytochemical techniques, 3,3'-dihexyloxa-carbocyanine iodide (DiOC6(3)) and calcofluor white in conjunction with computer-controlled video image analysis. In addition, confocal laser scanning microscopy has been used in conjunction with ethidium bromide to detect the nature and location of the sperm cell nuclear chromatin before and after karyogamy. Digitised video images of chlorotetracycline (CTC) fluorescence reveal that egg cells contain high levels of membrane Ca2+ in organelles present around the nucleus while the cytosolic signal is relatively low. Intense CTC fluorescence is invariably present just below the plasma membrane of egg cells and a certain degree of regionalised distribution of Ca2+ in cytoplasm is also discernible. Similarly, the fluphenazine (FPZ)-detectable calmodulin (CaM) and that localised immunocytochemically using monoclonal anti-CaM antibodies reveal high levels of CaM in the vicinity of the nucleus in egg cells. Only a few ER profiles and mitochondria could be visualised in the egg cell and no calcofluor fluorescence could be detected. Following in vitro fertilisation of single isolated eggs substantial changes in the Ca2+ levels occur which include an increase in the membrane Ca2+ of the fusion product, particularly in the cytosol and around the nucleus. Unlike in the eggs the fine CTC fluorescence signal below the plasma membrane is not detectable in the fusion products.(ABSTRACT TRUNCATED AT 250 WORDS)

Electro-fused isolated wheat (Triticum aestivum L.) gametes develop into multicellular structures.

The electrofusion-mediated fertilization of single egg cells of wheat with isolated individually selected wheat sperm cells was successfully carried out for the first time. On average the fusion frequency was 30% but under optimal conditions it was possible to reach as much as 55%. Two days after electric fusion 60% of the fusion products started to divide, 88.5% of them forming multicellular structures and in a few cases microcalluses. The culture of single unfertilized egg cells with or without the application of AC field and electric pulses induced no cell division. The egg cells and fusion products were cultured in a maize feeder-cell system.

In vitro fertilisation of maize by single egg and sperm cell protoplast fusion mediated by high calcium and high pH.

We present evidence for the fusion of isolated single maize egg and sperm cell protoplasts in a mannitol solution (400-430 mosmol/kg H2O) containing 0.05 M CaCl2 at pH 11.0, followed by cell division of the fusion products. These findings allow the performance of in vitro fertilisation of higher plants by combining single gametes as in lower plant and animal systems. Further, our findings open new avenues for investigating the basic mechanisms of adhesion and fusion of higher plant gametes and eventually for examining processes that inhibit polyspermy in higher plants.

Representative cDNA libraries from few plant cells.

A reverse transcriptase/polymerase chain reaction (RT/PCR) method for the construction of representative cDNA libraries originating from few isolated cells is described. Poly(A)+ RNA was extracted from an average of 100 maize cells and reverse transcribed into sscDNA. The sscDNA was dG-tailed at its 3' end and amplified during a two-step PCR reaction. The generated PCR products were analysed and the majority < or = 2 kbp were full-size cDNAs. A fraction of the amplified cDNA from 128 isolated maize egg cells was cloned into the lambda Uni-ZAP XR vector and a primary library of 6.8 x 10(6) p.f.u. was obtained. The average insert size is 860 bp. It was further determined, that 0.31% of the clones hybridized to a cytosolic GAPDH probe. It is thought that, with this method, the first cDNA library of egg cells in higher plants was generated.

In Vitro Fertilization with Isolated, Single Gametes Results in Zygotic Embryogenesis and Fertile Maize Plants.

We demonstrate here the possibility of regenerating phenotypically normal, fertile maize plants via in vitro fertilization of isolated, single sperm and egg cells mediated by electrofusion. The technique leads to the highly efficient formation of polar zygotes, globular structures, proembryos, and transition-phase embryos and to the formation of plants from individually cultured fusion products. Regeneration of plants occurs via embryogenesis and occasionally by polyembryony and organogenesis. Flowering plants can be obtained within 100 days of gamete fusion. Regenerated plants were studied by karyological and morphological analyses, and the segregation of kernel color was determined. The hybrid nature of the plants was confirmed.

Karyogamy after Electrofusion of Single Egg and Sperm Cell Protoplasts from Maize: Cytological Evidence and Time Course.

In maize, in vitro fusion of isolated male gametes with isolated egg cell protoplasts can be induced by electric pulses. Until now, karyogamy has not been demonstrated. In this study, we cytologically examined fusion products fixed at different times after electrofusion with phase contrast microscopy and transmission electron microscopy. We obtained a precise timetable from 23 samples studied during the first 3 hr. The sperm nucleus was integrated within the egg cell protoplast, migrated toward the egg cell nucleus, and fused with it within 1 hr, as demonstrated by ultrastructural observations, three-dimensional reconstructions of nuclei, and subsequent nuclear volume estimates. Fusion of nuclei occurred before zygotic mitosis, as is the case in vivo. These findings demonstrate karyogamy during in vitro fertilization of maize.

Analysis of single protoplasts and regenerated plants by PCR and RAPD technology.

We investigated the use of the polymerase chain reaction (PCR) and the associated random amplification of polymorphic DNA (RAPD) technique in the analysis of DNA and specific genes in plant cells at different stages of regeneration in in vitro cultures. We demonstrate that both procedures can be used to differentiate reproducibly between closely related species as well as to reveal levels of DNA polymorphism in regenerated plants. We also demonstrate that both procedures, using protocols that we have developed, are applicable at all tissue culture stages, from single isolated protoplasts to regenerated plants. Possible explanations for the variation levels detected in regenerated wheat plants are advanced.

Individual selection, culture and manipulation of higher plant cells.

Due to the heterogeneity in morphology, physiological and morphogenetical capabilities of higher plant cells in mass culture, the development of methods for individually culturing defined cells seemed to be useful and necessary. Individual cell culture represents a powerful tool for studies on the physiology of different cell types, the analysis of differentiation programs, the genetic manipulation of plant cells and cell-cell interactions. An improved microculture system based on a computer-controlled set-up for the efficient selection, transfer and individual culture of defined higher plant cells until regeneration of whole plants is described. Related experimental approaches for individually manipulating higher plant cells under controlled conditions, such as electrofusion of defined pairs of protoplasts and subprotoplasts, cell reconstruction and intranuclear microinjection of protoplasts and karyoplasts - mainly performed with cells of the crop plant Brassica napus L. - are presented.