Tobacco (Nicotiana tabacum L.) microspores at the time of mitosis are characterized by the abundant occurrence of 92- and 98-kDa glycoproteins (GP92 and GP98). GP92 is a soluble protein while GP98 is bound to the insoluble microspore fraction. Both glycoproteins were isolated by affinity chromatography and SDS-PAGE and analysed by MS. Peptide sequences were determined by mu-HPLC/nano-ESI-MS/MS (electrospray ionization tandem MS). GP92 displayed homology to beta-galactosidase (EC 3.2.1.23) and GP98 to beta-xylosidase (EC 3.2.1.37) from Arabidopsis thaliana (L.) Heynh. The activities of the two enzymes in microspore and pollen extracts of tobacco exhibited similar developmental changes to the occurrence of GP92 and GP98, with a maximum around microspore mitosis. These two glycoproteins are the first identified enzymes characteristic of mitotic microspores. Arabidopsis transcriptomic data for five beta-galactosidase and three beta-xylosidase genes abundantly expressed in pollen were verified by reverse transcription-PCR of RNA from different stages of Arabidopsis pollen development and from various parts of the sporophyte. The results showed abundant expression of two genes (At5g20710, At1g31740) homologous to tobacco GP92 in microspores and early pollen, and of three genes (At5g56870, At2g16730 and At4g35010) in maturing pollen. Analysis of beta-xylosidases showed abundant expression of a late pollen-specific gene At3g62710 and low expression of an early gene At5g10560. It is suggested that the early beta-galactosidase and beta-xylosidase genes may participate in cell wall loosening associated with pollen expansion after microspore mitosis and that the products of the late genes may play a role in cell expansion during pollen germination.

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Glycoproteins 50, 55, 59 and 64 kDa with affinity to the lectin ConA occurring abundantly in mature tobacco pollen were shown to exhibit high tolerance against heating at 90°C for 30°min. The 59 kDa glycoprotein (GP59) was isolated by affinity chromatography on ConA-agarose followed by 2D-electrophoresis and identified by MS analysis as tobacco calreticulin with approximate pI 4.2. Identification of the protein was confirmed by immunoblotting with human anti-calreticulin and by labelling with a specific dye for Ca 2+ -binding proteins (Stains All). Two acidic isoforms of 50 kDa glycoprotein in addition to GP59 displayed homology to calreticulin. RT-PCR revealed the presence of transcripts for calreticulin 59 kDa at all the stages of pollen development from microspore mitosis through the first 24 h of pollen tube growth. Immunodetection with anti-calreticulin and affinity to ConA on Western blots of total soluble proteins separated by 1D-SDS-PAGE showed that the protein first occurred at the mid-bicellular pollen stage, accumulated during pollen maturation and disappeared during 24 h of pollen tube growth. A thermotolerant form of GP59 was detected only in the terminal phase of pollen maturation and during 8 h of pollen tube growth. Results indicated that calreticulin 59 kDa is transcribed, translated and undergoes post-translational modification at a number of different stages of pollen development and that thermotolerance of the protein in mature pollen may be associated with high glycosylation. The thermotolerance of these glycoproteins could play a role in the protection of pollen against stress factors during dehydration and dispersal.