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Seed production generally requires the mating of opposite sex gametes. Apomixis, an asexual mode of reproduction, avoids both meiotic reduction and egg fertilization. The essential feature of apomixis is that an embryo is formed autonomously by parthenogenesis from an unreduced egg of an embryo sac generated through apomeiosis. If apomixis were well understood and harnessed, it could be exploited to indefinitely propagate superior hybrids or specific genotypes bearing complex gene sets. A more profound knowledge of the mechanisms that regulate reproductive events would contribute fundamentally to understanding the genetic control of the apomictic pathway. In Poa pratensis, we isolated and characterized two genes, PpSERK (SOMATIC EMBRYOGENESIS RECEPTOR-LIKE KINASE) and APOSTART. These full-length genes were recovered by rapid amplification of cDNA ends and their temporal and spatial expression patterns were assessed by reverse transcription-polymerase chain reaction and in situ hybridization, respectively. The expression of PpSERK and APOSTART differed in apomictic and sexual genotypes. Their putative role in cell-signaling transduction cascades and trafficking events required during sporogenesis, gametogenesis, and embryogenesis in plants is reported and discussed. We propose that, in nucellar cells of apomictic genotypes, PpSERK is the switch that channels embryo sac development and that it may also redirect signaling gene products to compartments other than their typical ones. The involvement of APOSTART in meiosis and programmed cell death is also discussed.

The expansins comprise a family of proteins that appear to be involved in the disruption of the noncovalent bonds between cellulose microfibrils and cross-linking glycans, thereby promoting wall creep. To understand better the expansion process in Petunia hybrida (petunia) flowers, we isolated a cDNA corresponding to the PhEXP1 alpha-expansin gene of P. hybrida. Evaluation of the tissue specificity and temporal expression pattern demonstrated that PhEXP1 is preferentially expressed in petal limbs during development. To determine the function of PhEXP1, we used a transgenic antisense approach, which was found to cause a decrease in petal limb size, a reduction in the epidermal cell area, and alterations in cell wall morphology and composition. The diminished cell wall thickness accompanied by a reduction in crystalline cellulose indicates that the activity of PhEXP1 is associated with cellulose metabolism. Our results suggest that expansins play a role in the assembly of the cell wall by affecting either cellulose synthesis or deposition.

The smaller isoform of the enzyme glutamic acid decarboxylase (GAD65) is a major islet autoantigen in autoimmune type 1 diabetes mellitus (T1DM). Transgenic plants expressing human GAD65 (hGAD65) are a potential means of direct oral administration of the islet autoantigen in order to induce tolerance and prevent clinical onset of disease. We have previously reported the successful generation of transgenic tobacco and carrot that express immunoreactive, full-length hGAD65. In the present study, we tested the hypothesis that the expression levels of recombinant hGAD65 in transgenic plants can be increased by targeting the enzyme to the plant cell cytosol and by mediating expression through the potato virus X (PVX) vector. By substituting the NH2-terminal region of hGAD65 with a homologous region of rat GAD67, a chimeric GAD67(1-87)/GAD65(88-585) molecule was expressed in transgenic tobacco plants. Immunolocalization analysis showed that immunoreactive GAD67/65 was found in the plant cell cytosol. By using a radio-immuno assay with human serum from a GAD65 autoantibody-positive T1DM patient, the highest expression level of the recombinant GAD67/65 protein was estimated to be 0.19% of the total soluble protein, compared to only 0.04% of wild-type hGAD65. Transient expression of wild-type, full-length hGAD65 in N. benthamiana mediated by PVX infection was associated with expression levels of immunoreactive protein as high as 2.2% of total soluble protein. This substantial improvement of the expression of hGAD65 in plants paves the way for immunoprevention studies of oral administration of GAD65-containing transgenic plant material in animal models of spontaneous autoimmune diabetes.