Agrobacterium-mediated transformation of cereals: a promising approach crossing barriers.

Cereal crops have been the primary targets for improvement by genetic transformation because of their worldwide importance for human consumption. For a long time, many of these important cereals were difficult to genetically engineer, mainly as a result of their inherent limitations associated with the resistance to Agrobacterium infection and their recalcitrance to in vitro regeneration. The delivery of foreign genes to rice plants via Agrobacterium tumefaciens has now become a routine technique. However, there are still serious handicaps with Agrobacterium-mediated transformation of other major cereals. In this paper, we review the pioneering efforts, existing problems and future prospects of Agrobacterium-mediated genetic transformation of major cereal crops, such as rice, maize, wheat, barley, sorghum and sugarcane.

A possible role of sphingolipids in the aluminium resistance of yeast and maize.

The plasma membrane is most likely the major target for sensing of aluminium (Al), leading to inhibition of plant root-growth. As a result of high external Al, alterations in plasma membrane composition may be expected in order to maintain its properties. As sphingolipids are characteristic components of this membrane, their involvement in membrane adjustment to increased Al concentrations was investigated. Heterologous expression of a stereounselective long-chain base (LCB) (8E/Z)-desaturase from Arabidopsis thaliana, Brassica napus and Helianthus annuus in Saccharomyces cerevisiae improved the Al resistance of the transgenic yeast cells. This encouraged us to investigate whether Al affects the LCB composition, and whether genetic engineering of the LCB profile modifies the Al resistance of the Al-sensitive plant species maize (Zea mays, L.). Constitutive expression of the LCB (8E/Z)-desaturase from Arabidopsis thaliana in maize roots led to an 8- to 10-fold increase in (8E)-4-hydroxysphing-8-enine in total roots. Less marked but similar changes were observed in 3 mm root apices. Al treatment of the Al-sensitive maize cv Lixis resulted in a significant increase in the proportion of (8Z)-LCB and in the content of total LCBs in root tips, which was not observed in the Al-resistant cv ATP-Y. When root tips of transgenic plants were exposed to Al, only minor changes of both (8Z)- and (8E)-unsaturated LCBs as well as of the total LCB were observed. Al treatment of the wild type parental line H99 decreased the (8Z)-unsaturated LCBs and the total LCB content. Based on Al-induced callose production, a marker for Al sensitivity, the parental line H99 was as Al-resistant as cv ATP-Y, whereas the transgenic line became as sensitive as cv Lixis. Taken together, these data suggest that, in particular, the loss of the ability to down-regulate the proportion of (8Z)-unsaturated LCBs may be related to increased Al sensitivity.

Expression of transgenic stilbene synthases in wheat causes the accumulation of unknown stilbene derivatives with antifungal activity.

The expression of foreign phytoalexins in a new host is thought to increase fungal resistance, since host-specific pathogens have not experienced selection for detoxifying or metabolising the novel antifungal compounds. Two resveratrol synthase genes vst1 and vst2 from grapevine (Vitis vinifera L.) and the pinosylvin synthase gene pss from pine (Pinus sylvestris L.) were stably transformed into bread wheat. The expression of the target genes is regulated by stress-inducible grapevine promoters. The vst1 and vst2 promoters were functional in wheat and retained their expression profiles described for grapevine. ALL vst and pss transgenic lines accumulated stilbene derivatives upon induction by UV light. The detected stilbenes showed a remarkable similarity to resveratrol and pinosylvin, however were found to be more hydrophilic than resveratrol and pinosylvin. Upon inoculation with the biotrophic pathogen Puccinia recondita f.sp. tritici several vst expressing wheat lines showed a significant reduction of disease symptoms (19 +/- 9% to 27 +/- 8%) compared to wild-type plants. The reduction of disease symptoms was even more obvious after inoculation with the facultative biotrophic pathogen Septoria nodorum Berk. and ranged from 42 +/- 13% to 71 +/- 4%. None of the four tested pss expressing lines showed a reduction in disease incidence.

Rust and downy mildew resistance in pearl millet (Pennisetum glaucum) mediated by heterologous expression of the afp gene from Aspergillus giganteus.

The cDNA encoding the antifungal protein AFP from the mould Aspergillus giganteus was introduced into two pearl millet (Pennisetum glaucum) genotypes by particle bombardment. Stable integration and expression of the afp gene was confirmed in two independent transgenic T0 plants and their progeny using Southern blot and RT-PCR analysis. In vitro infection of detached leaves and in vivo inoculation of whole plants with the basidomycete Puccinia substriata, the causal agent of rust disease, and the oomycete Sclerospora graminicola, causal agent of downy mildew, resulted in a significant reduction of disease symptoms in comparison to wild type control plants. The disease resistance of pearl millet was increased by up to 90% when infected with two diverse, economically important pathogens. This is the first report of genetic enhancement of Pennisetum glaucum against fungal infections.

Gynogenic plant regeneration from unpollinated flower explants of Eragrostis tef (Zuccagni) Trotter.

Tef [Eragrostis tef (Zucc.) Trotter] is the most important cereal in Ethiopia. In its wild relative E. mexicana, regeneration of six green plants resulted from culture of 121 non-pollinated immature pistils. In the allotetraploid crop species tef, however, only callus and root formation was obtained by this method. By contrast, immature spikelets and panicle segments of E. tef proved amenable to gynogenic plant regeneration. Upon step-wise optimization of the protocol, efficient plant formation was achieved in all three cultivars tested. In cv. DZ-01-196, culture of 1305 immature spikelets resulted in formation of 159 green plants. Flow cytometric analysis revealed (di)haploid, triploid, tetraploid and octoploid regenerants, from which the vast majority was tetraploid. Tef-breeding programs will likely benefit substantially from efficient generation of true-breeding plants.

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.

WM5: Isolation and characterisation of a gene expressed during early meiosis and shoot meristem development in wheat.

Wheat Meiosis 5 (WM5), isolated from an early meiosis anther cDNA library of wheat by cDNA subtraction encodes a novel glycine-serine-proline-alanine-rich protein. The corresponding homologous genes are located on the short arms of chromosomes 3A, 3B and 3D of allohexaploid wheat (Triticum aestivum L.). The copy on 3DS is located within the region deleted in the wheat mutant ph2a that displays increased homoeologous chromosome pairing in crosses with alien species. While WM5 is expressed primarily in young flower buds during early meiosis it is also expressed in shoot meristems, thus indicating functional roles in both meiosis and meristem development. Overall, the WM5 amino acid sequence shares no significant similarity with other known proteins in the NCBI database. However, the carboxyl-terminal region does have similarity with the Arabidopsis PDF1 (Protodermal Factor 1) protein. Comparing WM5 and PDF1 reveals that the two proteins share 33% identity and have similar hydropathy plots and predicted secondary structures. In situ immuno-staining locates the protein to the nuclei of pollen mother cells undergoing meiosis and the epidermal layer of the shoot and flower meristem, including the cell wall and cuticle. We propose that the WM5 protein has a role in shoot and flower development within this economically important cereal crop.

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.

The sugary-type isoamylase in wheat: tissue distribution and subcellular localisation.

To gain an increased understanding of the role of isoamylase (EC 3.2.1.68) in amylopectin synthesis, we studied the tissue-specific distribution and subcellular localisation of this enzyme in wheat (Triticum aestivum L.). A cDNA for wheat isoamylase was isolated from an endosperm-specific library and the missing 5' end was amplified by anchored polymerase chain reaction. Isoamylase transcripts were detected in reproductive and vegetative tissues, with the highest levels occurring in developing kernels. Wheat kernels were then dissected into embryo, endosperm, pericarp and chlorophyll layer, and subjected to protein blot analysis. Isoamylase was most abundant in the endosperm. Within the endosperm, the vast majority of isoamylase was soluble. A much smaller amount of the enzyme was associated with starch granules. Isoamylase was not trapped within starch granules and was absent from dry seeds. Isoamylase was also present in green tissue, which suggests a role in the synthesis of both reserve and leaf starches.

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.

5' deletion of a gbss1 promoter region from wheat leads to changes in tissue and developmental specificities.

Expression of granule-bound starch synthase 1 (GBSS1) in wheat is restricted to the grain filling process. In order to identify promoter regions which are involved in transcriptional control of the observed expression pattern, we isolated about 8 kb of a wheat gbss1-upstream region. Within this sequence several putative cis-acting elements were identified. In addition, an untranslated leader region is located in the 5' region of the gbss1 gene. To investigate promoter activity of the isolated region, the proximal 4.0 kb and progressively 5'-deleted fragments were transcriptionally fused to a beta-glucuronidase reporter gene. The function of the promoter constructs was tested by transient expression assays in various wheat tissues and in transgenic wheat plants, which were selected for low number and integrity of transgene copies. Analysis of stable transformants revealed that the -4.0 kb promoter region mediates reporter gene expression that is in accordance with the endogenous gbss1 expression. Promoter deletion to -1.9 kb or to -1.0 kb did not change the expression profile with regard to grain and pollen specificity. However, the profile of beta-glucuronidase expression during the grain filling process is altered in such a way that the level of beta-glucuronidase activity declines due to the decreasing promoter length. It is proposed that enhancer elements and cis-acting elements, which are involved in gbss1 transcription during the grain filling process, are located -1.9 kb upstream of the promoter. In addition, participation of the untranslated leader region in tissue-specific gene expression is discussed.

A novel nucleus-targeted protein is expressed in barley leaves during senescence and pathogen infection.

The barley (Hordeum vulgare) cDNA HvS40 represents a gene with enhanced mRNA level during leaf senescence. Biolistic transformation of onion (Allium cepa) epidermal cell layers with a glucuronidase fusion protein construct provided evidence that the 15.4-kD protein encoded by HvS40 is localized to the nucleus. Expression of the gene is induced by jasmonate and salicylic acid; both are known to act as signaling compounds during senescence and defense toward pathogens. Transcript levels of HvS40 were observed to be particularly high in leaf sectors that undergo necrosis and chlorosis after infection with Pyrenophora teres. This pathogen-related expression is, in contrast, clearly reduced in transgenic barley plants expressing a stilbene synthase from grape (Vitis vinifera), whereas the mRNA level of a gene encoding the pathogen-related protein HvPR1 is unaffected. In situ hybridization with HvS40 antisense RNA revealed that during leaf senescence, the HvS40 transcript predominantly accumulates in the mesophyll. Taken together, the findings suggest a connection between the nuclear protein encoded by HvS40 and the degeneration of chloroplasts occurring during senescence and during infection of barley wild-type plants with P. teres.