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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 GAD671-87/GAD6588-585molecule 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. benthamianamediated 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.

The definition of the patterns of cell division and expansion in plant development is of fundamental importance in understanding the mechanics of morphogenesis. By studying cell division and expansion patterns, we have assembled a developmental map of Petunia hybrida petals. Cycling cells were labelled with in situ markers of the cell cycle, whereas cell expansion was followed by assessing cell size in representative regions of developing petals. The outlined cell division and expansion patterns were related to organ asymmetry. Initially, cell divisions are uniformly distributed throughout the petal and decline gradually, starting from the basal part, to form a striking gradient of acropetal polarity. Cell areas, in contrast, increased first in the basal portion and then gradually towards the petal tip. This growth strategy highlighted a cell size control model based on cell-cycle departure time. The dorso-ventral asymmetry can be explained in terms of differential regulation of cell expansion. Cells of the abaxial epidermis enlarged earlier to a higher final extent than those of the adaxial epidermis. Epidermal appendage differentiation contributed to the remaining asymmetry. On the whole our study provides a sound basis for mutant analyses and to investigate the impact of specific (environmental) factors on petal growth.

Mutants showing features of apomixis have been documented in alfalfa (Medicago sativaL.), a natural outcrossing sexual species. A differential display of mRNAs that combines cDNA-AFLP markers and bulked segregant analysis was carried out with the aim of selecting expressed sequence tags (ESTs) and cloning candidate genes for apomeiosis in mutants of alfalfa characterized by 2negg formation at high frequencies. The approach enabled us to select either mutant- or wild type-specific transcript derived-fragments and to detect transcriptional changes potentially related to 2neggs. Sequence alignments of a subset of 40 polymorphic clones showed significant homologies to genes of known function. An EST with identity to a β-tubulin gene, highly expressed in the wild type and poorly expressed in the apomeiotic mutants, and an EST with identity to a Mob1-like gene, qualitatively polymorphic between pre- and post-meiotic stages, were selected as candidate genes for apomeiosis because of their putative roles in the cell cycle. A number of clone-specific primers were designed for performing both 5′ and 3′ rapid amplification of cDNA ends to obtain the full-length clones. Southern blot hybridization revealed that both clones belong to a multi-gene family with a minimum of three genomic DNA members each. Northern blot hybridization of total RNA samples and in situ hybridization of whole buds enabled the definition of their temporal and spatial expression patterns in reproductive organs. Experimental achievements towards the elucidation of apomeiotic megasporogenesis in alfalfa are presented and discussed

Despite the potential that apomixis has for agriculture, there is little information regarding the genetic control of its functional components. We carried out a cytohistological investigation on an F1segregating population ofPoa pratensisobtained from a cross between a sexual and an apomictic parent. About half of the F1progeny plants were parthenogenic, as adjudicated by an auxin test. The degree of parthenogenesis ranged from 1.44% to 92.9%. Apospory was detected in parthenogenetic plants as well as in two non-parthenogenetic individuals. These results indicate that two distinct genetic factors control apospory and parthenogenesis inP. pratensisand that apospory and parthenogenesis may be developmentally uncoupled

Gametophytic apomixis in Kentucky bluegrass (Poa pratensisL.) involves the parthenogenetic development of unreduced eggs from aposporic embryo sacs. Marker-assisted selection for the mode of reproduction in P. pratensiswould avoid costly and time-consuming phenotypic progeny tests. We developed and tested two SCAR primer pairs that are associated with the mode of reproduction in P. pratensis. The SCAR primers identified the apomictic and sexual genotypes among progenies of sexual x apomictic crosses with very low bias. Furthermore, when tested on a wide range of Italian and exotic P. pratensisgermplasm, they were able to unequivocally distinguish sexual from apomictic genotypes. This system should, therefore, allow new selection models to be set up in this species.

Cyclin-dependent kinases (CDKs) control the key transitions in the eukaryotic cell cycle. All the CDKs known to control G2/M progression in yeast and animals are distinguished by the characteristic PSTAIRE motif in their cyclin-binding domain and are closely related. Higher plants contain in addition a number of more divergent non-PSTAIRE CDKs with still obscure functions. We show that a plant-specific type of non-PSTAIRE CDKs is involved in the control of the G2/M progression. In synchronized tobacco BY-2 cells, the corresponding protein, accumulated in a cell cycle-regulated fashion, peaking at the G2/M transition. The associated histone H1 kinase activity reached a maximum in mitosis and required a yet unidentified subunit to be fully active. Down-regulation of the associated kinase activity in transgenic tobacco plants using a dominant-negative mutation delayed G2/M transition. These results provide the first evidence that non-PSTAIRE CDKs are involved in the control of the G2/M progression in plants.

A cyclin cDNA clone (Pethy;CycB1;1) was isolated from a Petunia hybridaovary specific cDNA library. Sequence comparison revealed that Pethy;CYCB1;1 protein is highly homologous to mitotic B1 cyclins. Northern analysis and in situ hybridisation experiments showed that its expression is developmentally regulated and restricted to flower organs. We have attempted to define some of the cell division patterns which contribute to shaping each floral organ by analysing Pethy;CycB1;1expression on Petuniaflower sections. While in sepals, epidermis and parenchyma cell division patterns were comparable, there were two distinct cell division patterns in petals. In the epidermis, Pethy;CYCB1;1expression was found both at the petal tip and along epidermis, whereas in the parenchyma only at the petal tips. In reproductive organs cell divisions were detected only in sporophytic tissues. No signals were detected inside meiotic cells.

A cyclin cDNA clone ( Pethy;CycB1;1) was isolated from a Petunia hybridaovary specific cDNA library. Sequence comparison revealed that Pethy;CYCB1;1 protein is highly homologous to mitotic B1 cyclins. Northern analysis and in situ hybridisation experiments showed that its expression is developmentally regulated and restricted to flower organs. We have attempted to define some of the cell division patterns which contribute to shaping each floral organ by analysing Pethy;CycB1;1expression on Petuniaflower sections. While in sepals, epidermis and parenchyma cell division patterns were comparable, there were two distinct cell division patterns in petals. In the epidermis, Pethy;CYCB1;1expression was found both at the petal tip and along epidermis, whereas in the parenchyma only at the petal tips. In reproductive organs cell divisions were detected only in sporophytic tissues. No signals were detected inside meiotic cells.

Parenteral and oral administration of autoantigens can induce immune tolerance in autoimmune diseases. Prophylactic therapy based on oral administration of human autoantigens is not, however, feasible when sufficient quantities of candidate autoantigens are not available. Transgenic plants that express high levels of recombinant proteins would allow large quantities of autoantigens to be produced at relatively low costs. In addition, transgenic food would provide a simple and direct method of delivering autoantigens. The production and the characterization of transgenic tobacco and carrot plants expressing human GAD65, a major autoantigen in human insulin-dependent diabetes mellitus (IDDM), is reported. Immunogold labeling and electron microscopy of transgenic tobacco tissue shows the selective targeting of human GAD65 to chloroplast tylacoids and mitochondria. In planta expressed GAD65 has a correct immunoreactivity with IDDM-associated autoantibodies and retains enzymatic activity, a finding that suggests a correct protein folding. In transgenic tobacco and carrot the expression levels of human GAD65 varies between 0.01% and 0.04% of total soluble proteins. Transgenic edible plant organs are now available to study the feasibility of inducing immune tolerance in IDDM animals by oral administration of GAD65.