Abstract
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 Ca2+ -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.
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Acknowledgements
This research was supported by grant 522/02/D075 of the Grant Agency of the Czech Republic (PH), grant 1K03018 of the Ministry of Education of the Czech Republic (DH) and grant B6038409 of the Grant Agency of the Academy of Sciences of the Czech Republic (DH).
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Hrubá, P., Honys, D. & Tupý, J. Expression and thermotolerance of calreticulin during pollen development in tobacco. Sex Plant Reprod 18, 143–148 (2005). https://doi.org/10.1007/s00497-005-0007-z
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DOI: https://doi.org/10.1007/s00497-005-0007-z