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We used electron microscopical techniques to study ultrastructural changes during the acquisition of embryogenic competence in immature pollen grains ofNicotiana tabacum, isolated at the early- or mid-bicellular stage and cultured in vitro under starvation conditions. Cytoplasmic and nuclear changes during the starvation treatment are reported. Dedifferentiation of plastids, dilation of the wall of the generative cell, the appearance of a large vacuole, loss of nuclear pores in the vegetative nucleus, changes in chromatin and nucleolar structure, and a decrease in the size of the nucleolus were observed. We suggest that these events are the first step in the switch from generative to vegetative generation during pollen embryogenesis.

Bet v 1 proteins, a group of isoforms that constitute the major pollen allergen of the white birch (Betula verrucosa), are encoded by a complex multigene family which also includes a subset of genes transcriptionally activated in cell suspension cultures in the presence of microbial pathogens. We have investigated the temporal and spatial expression patterns ofBet v 1 genes during development of male birch inflorescences. Western and Northern analysis indicated that the expression of Bet v 1 isoallergens is regulated at the transcriptional level and activated only during the late phases of anther maturation, so that high levels of these proteins accumulate in mature pollen. In situ hybridization experiments, carried out with sections of male inflorescences isolated at defined developmental stages, showed thatBet v 1 genes are specifically expressed in late bicellular and mature pollen, but no transcripts were detected in sporophytic anther tissues of any developmental stage. These data suggest a possible role of Bet v 1 proteins during pollen germination on the stigma, protecting the female reproductive tissues from infection by pathogens.

Two novel beta-expansin genes, TaEXPB1 and TaEXPB2, were isolated from wheat microspores by suppression subtractive hybridisation. Northern blot and reverse transcription PCR analyses showed that the expression of both genes was restricted to early stages of male gametophyte development (from microspores to immature pollen). A homology search showed high similarity of the newly discovered genes to generative beta-expansins in grass pollen (group 1 pollen allergens). Southern hybridisation revealed that the isolated genes belong to a distinct group within the subfamily of beta-expansin genes in the wheat genome. A comparison of full-length cDNAs with the corresponding genomic sequences showed that there are two introns in the TaEXPB1 gene, whereas TaEXPB2 has three introns. Both genes were predicted to encode highly similar basic proteins (pI 9.0) with molecular masses of approximately 29 kDa consisting of a signal peptide, catalytic, and polysaccharide binding domains, which include conserved cysteines and tryptophans and motifs characteristic for beta-expansins.