L.J.W.J. Gilissen

Genomic cloning and linkage mapping of the Mal d 1 (PR-10) gene family in apple (Malus domestica).

Fresh apples can cause birch pollen-related food allergy in northern and central European populations, primarily because of the presence of Mal d 1, the major apple allergen that is cross-reactive to the homologous and sensitizing allergen Bet v 1 from birch. Apple cultivars differ significantly in their allergenicity. Knowledge of the genetic basis of these differences would direct breeding for hypoallergenic cultivars. The PCR genomic cloning and sequencing were performed on two cultivars, Prima and Fiesta, which resulted in 37 different Mal d 1 gDNA sequences. Based on the mapping of sequence-specific molecular markers, these sequences appeared to represent 18 Mal d 1 genes. Sixteen genes were located in two clusters, one cluster with seven genes on linkage group (LG) 13, and the other cluster with nine genes on the homoeologous LG 16. One gene was mapped on LG 6, and one remained unmapped. According to sequence identity, these 18 genes could be subdivided into four subfamilies. Subfamilies I–III had an intron of different size that was subfamily and gene-specific. Subfamily IV consisted of 11 intronless genes. The deduced amino acid sequence identity varied from 65% to 81% among subfamilies, from 82% to 100% among genes within a subfamily, and from 97.5% to 100% among alleles of one gene. This study provides a better understanding of the genetics of Mal d 1 and the basis for further research on the occurrence of allelic diversity among cultivars in relation to allergenicity and their biological functions.

Development of polysomaty during differentiation in diploid and tetraploid tomato (Lycopersicon esculentum) plants

Abstract Polysomaty was found in all organs of tomato (Lycopersicon esculentum cv. Moneymaker) tested. The first endoreplications occurred in cotyledon and hypocotyl during germination (concurrent with cell elongation). Also the stem and leaves became polysomatic during development (cell elongation and expansion). Finally, new rounds of endoreplications marked the yellowing of the leaves (ageing), during which, in the leaf petiole, levels up to 128C were reached (1C is the DNA content of germ-line cells). Comparing diploid and tetraploid plants, the patterns of polysomaty were similar in four organs tested. The frequency of nuclei at the different C-levels in diploid cotyledons (2C, 51.6% of the nuclei; 4C, 39.0%; 8C, 8.6%; 16C, 0.8%) equalled the corresponding levels in tetraploid cotyledons (2C, 52.1%; 4C, 37.2%; 8C, 9.6%; 16C, 0.9%). Differences in growth and leaf expansion between in vitro- and greenhouse-grown plants were reflected in differences in polysomaty patterns between corresponding cotyledon and leaf tissues. These results indicate that polysomaty is genetically regulated as the number of endoreduplications taking place in the cells, and influenced by the actual growth pattern of the plant. As such, it may be considered as an integral part of the morphogenesis of a plant, from germination to ageing.

Development of polysomaty in seedlings and plants of Cucumis sativus L.

Abstract The nuclear DNA content of cells from individual organs of seedlings and plants of Cucumis sativus L. (cv. Hokus) was determined during development by flow cytometry. The results revealed the presence of cells with different DNA contents (ranging from 2C to 64C) in varying proportions. This polysomaty was both specific to the developmental stage of the plant and to the individual organ. Polysomaty was already present in the hypocotyl and radicle of the embryo in the seed, and also in young secondary roots, immature leaves, flower buds and very young stem internodes. Rapid increases of the nuclear DNA contents (endopolyploidy) and the number of C-value levels occurred in all organs of the seedling and in the stem internodes and flowers during elongation. In the seedlings, these increases originated in the transition tissue between hypocotyl and radicle. In the leaves the initial pattern of polysomaty did not change during expansion. Compared to other organs, the leaves showed the highest frequency (60%) of cells with a 2C DNA content. During plant maturation, the proportion of nuclei with higher DNA contents gradually increased in the radicle, the hypocotyl and the stem internodes. The results are discussed in relation to organ elongation, to genome size and culture conditions, and to their relevance to molecular and cellular approaches for cultivar improvement.

Genomic characterization and linkage mapping of the apple allergen genes Mal d 2 (thaumatin-like protein) and Mal d 4 (profilin)

Four classes of apple allergens (Mal d 1, −2, −3 and −4) have been reported. By using PCR cloning and sequencing approaches, we obtained genomic sequences of Mal d 2 (thaumatin-like protein) and Mal d 4 (profilin) from the cvs Prima and Fiesta, the two parents of a European reference mapping population. Two copies of the Mal d 2 gene (Mal d 2.01A and Mal d 2.01B) were identified, which primarily differed in the length of a single intron (378 or 380 nt) and in one amino acid in the signal peptide. Both Mal d 2.01A and Mal d 2.01B were mapped at identical position on linkage group 9. Genomic characterization of four Mal d 4 genes (Mal d 4.01A and B, Mal d 4.02A and Mal d 4.03A) revealed their complete gDNA sequences which varied among genes in length from 862 to 2017 nt. They all contained three exons of conserved length: 123, 138, and 135 nt. Mal d 4.01 appeared to be duplicated in two copies and located on linkage group 9. Mal d 4.02A and Mal d 4.03A were single copy genes located on linkage group 2 and 8, respectively.

The influence of perceived benefits on acceptance of GM applications for allergy prevention

Allergic diseases, such as hay fever and food allergy, affect an increasing part of the population in Westernized countries and have a negative impact on the patients quality of life. Allergy prevention measures that focus on reducing the allergenic load are currently developed, and these may include the use of genetic modification of allergenic plants. Such developments should take societal concerns about genetic modification into account. We examined the attitude of allergic and non-allergic respondents towards applications of genetic modification for allergy prevention in one food allergy application (apple) and two hay fever applications (birch, grass). Attitude towards genetic modification was described in terms of ‘benefits’ and ‘rejection factors.’ We found that respondents suffering from self-reported allergy perceived greater benefits associated with the birch application as compared to non-sufferers. The perceived benefits increased with an increasing impact of allergic complaints on quality of life. No differences were found between sufferers and non-sufferers for the food allergy application. The impact of perceived benefits on acceptance was larger than that of rejection factors. This supports the idea that acceptance of genetic modification is primarily a function of perceived personal benefit. Novel genetically modified products that are perceived to be beneficial by some consumers may consequently experience an increased consumer acceptance.

Detailed Analysis of the Expression of an Alpha-gliadin Promoter and the Deposition of Alpha-gliadin Protein During Wheat Grain Development

BACKGROUND AND AIMSnAlpha-gliadin proteins are important for the industrial quality of bread wheat flour, but they also contain many epitopes that can trigger celiac (coeliac) disease (CD). The B-genome-encoded alpha-gliadin genes, however, contain very few epitopes. Controlling alpha-gliadin gene expression in wheat requires knowledge on the processes of expression and deposition of alpha-gliadin protein during wheat grain development.nnnMETHODSnA 592-bp fragment of the promotor of a B-genome-encoded alpha-gliadin gene driving the expression of a GUS reporter gene was transformed into wheat. A large number of transgenic lines were used for data collection. GUS staining was used to determine GUS expression during wheat kernel development, and immunogold labelling and tissue printing followed by staining with an alpha-gliadin-specific antibody was used to detect alpha-gliadin protein deposited in developing wheat kernels. The promoter sequence was screened for regulatory motifs and compared to other available alpha-gliadin promoter sequences.nnnKEY RESULTSnGUS expression was detected primarily in the cells of the starchy endosperm, notably in the subaleurone layer but also in the aleurone layer. The alpha-gliadin promoter was active from 11 days after anthesis (DAA) until maturity, with an expression similar to that of a 326-bp low molecular weight (LMW) subunit gene promoter reported previously. An alpha-gliadin-specific antibody detected alpha-gliadin protein in protein bodies in the starchy endosperm and in the subaleurone layer but, in contrast to the promoter activity, no alpha-gliadin was detected in the aleurone cell layer. Sequence comparison showed differences in regulatory elements between the promoters of alpha-gliadin genes originating from different genomes (A and B) of bread wheat both in the region used here and upstream.nnnCONCLUSIONSnThe results suggest that additional regulator elements upstream of the promoter region used may specifically repress expression in the aleurone cell layer. Observed differences in expression regulator motifs between the alpha-gliadin genes on the different genomes (A and B) of bread wheat leads to a better understanding how alpha-gliadin expression can be controlled.

The competence of cells for cell division and regeneration in tobacco explants depends on cellular location, cell cycle phase and ploidy level

Abstract This work concerns application of flow cytometry (FCM) and confocal laser scanning microscopy (CLSM) to investigate the competence of cells for cell division and regeneration. FCM analysis of freshly-cut thin cell layer (TCL) explants of Nicotiana tabacum , excised from upper internodes of vegetative plants, revealed that one-quarter of the cells had the 2C nuclear DNA content, whereas of the other cells most nuclei had the 4C and some had the 8C DNA content. Cytometric examination using CLSM showed that the 2C nuclei were mainly located in the epidermis and subepidermis, and the 4C nuclei predominantly in the cortical tissue. During culture of the explants, part of the cortical cells went through mitosis from the first day onwards, and formed callus from which predominantly diploid and some tetraploid roots regenerated at low frequency. Most cortical cells were thus in the G 2 phase of the diploid cell cycle. FCM analysis showed that another fraction of the 4C cortical cells was induced to endoreduplicate to 8C cells. These cells thus had previously switched to the G 1 phase of the tetraploid cell cycle. CLSM analysis revealed that subepidermal and epidermal cells, respectively, underwent cell division from the second and third day onwards. Shoot primordia developed from cells of both cell layers together. Most shoot regenerants were normal diploids, some were mixoploids or tetraploids. The combination of FCM and CLSM allowed identification of the cell cycle phase, the ploidy level, the position of the cell, and the cellular development. The results give insight into the involvement of these parameters in the competence for cell division and regeneration at the level of the individual explant cells, and are therefore relevant for cellular and molecular approaches to plant transformation.

Somatic hybridization between potato and Nicotiana plumbaginifolia

SummaryElectrofusion was carried out between mesophyll protoplasts from the transformed diploid S. tuberosum clone 413 (2n=2x=24) which contains various genetic markers (hormone autotrophy, opine synthesis, kanamycin resistance, β-glucuronidase activity) and mesophyll protoplasts of a diploid wild-type clone of N. plumbaginifolia (2n=2x=20). Hybrid calli were obtained after continuous culture on selection medium containing kanamycin. Parental chromosome numbers, determined at 2 months after fusion, revealed hybrid-specific differences between the individual calli. On the basis of these differences three categories of hybrids were distinguished. Category I hybrids contained between 8 and 24 potato chromosomes and more than 20 N. plumbaginifolia chromosomes; category II hybrids had between 1 and 20 N. plumbaginifolia chromosomes and more than 24 potato chromosomes; category III hybrids contained diploid or subdiploid numbers of chromosomes from both parents. The hybrids were evenly distributed over the three categories. After a 1-year culture of 24 representative hybrid callus lines on selection medium the karyotype of 10 hybrids remained stable, whereas 8 hybrids showed polyploidization of the genome of one parent, together with no or minor changes of the chromosome numbers of the other parent. Six hybrids showed slight changes in the hybrid karyotype. The elimination of chromosomes of a particular parent was not correlated to their metaphase location. The processes of spontaneous biparental chromosome elimination leading to the production of asymmetric hybrids of different categories are discussed.

Natural variation in patterns of polysomaty among individual tomato plants and their regenerated progeny

Abstract Using flow cytometry, the patterns of polysomaty were determined in individual organs of seedlings of tomato ( Lycopersicon esculentum L. cv. Moneymaker). On average, each organ (i.e. leaf, cotyledon, transition zone from hypocotyl to primary root, and secondary root) displayed its own pattern of polysomaty. However, the percentages of nuclei at each C-level (1C = the DNA content after meiosis) varied greatly among individual plants, notably in cotyledons and transition zones. In the latter organ, the percentages of 2C-nuclei ranged from 19 to 43%. When plotted against the percentage of 2C-nuclei, the percentages of nuclei at other C-levels in leaf, cotyledon and transition zone fitted in a continuous pattern. In agreement with this, the polysomaty patterns of some leaves with relatively high levels of polysomaty were identical to those of the cotyledons with relatively low levels of polysomaty. The same was the case with some cotyledon and transition zone samples. These results might suggest developmental variation among the individual organs. However, the polysomaty patterns of individual cotyledons did not correlate with other parameters of development, such as the size of the organ, nor with the patterns of polysomaty in the leaf or transition zone of the same seedling. In first generation seedlings from plants regenerated from tissue culture, the extent of the variation in polysomaty had increased, especially in the cotyledons. Also, seedling populations of several regenerants displayed aberrant frequency distributions of 2C-nuclei in the cotyledons. However, the second-generation progeny did not show any of these aberrations, and the standard deviations of the second generation cotyledons were comparable to those of the control population. It is concluded that the deviations in the pattern of polysomaty can be due to unstable, epigenetic changes. The results are discussed in relation to the plasticity of the development and differentiation of individual organs.

First successful reduction of clinical allergenicity of food by genetic modification: Mal d 1 silenced apples cause fewer allergy symptoms than the wild-type cultivar

Genetic modification of allergenic foods such as apple has the potential to reduce their clinical allergenicity, but this has never been studied by oral challenges in allergic individuals.