Expression and thermotolerance of calreticulin during pollen development in tobacco

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 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.

Protein Changes during Heat Stress in Three Kentucky Bluegrass Cultivars Differing in Heat Tolerance

“…Western blotting showed that the 64 kDa polypeptide is a member of the HSP60 family (Singh and Lakhotia, 2000). Glycoproteins 64 kDa with affinity to the lectin ConA may be involved in pollen heat tolerance in tobacco ( Nicotiana tabacum L.) (Hrubá et al, 2005). It has been reported that 42 kDa proteins induced in the cytoplasm in Saccharomyces cerevisiae form ordered oligomers with a barrel‐like structure and act as a chaperone either in vivo or in vitro, which suppressed the aggregation of one‐third of the cytosolic proteins ((Haslbeck et al, 2004; Haslbeck, 2006).…”

Calcium function and distribution during fertilization in angiosperms

“…Presumably, a wider examination of expressed transcripts and proteomic data in pollen (Hruba et al, 2005;Sheoran et al, 2006) and other tissues (Swanson et al, 2005) may aid in identifying specific protein candidates for such calcium binding; information about such products is likely to be readily available for bioinformatic data mining in international genome and proteome related databases. This calcium store is dynamic and may therefore be readily converted to (and from) free calcium through interaction with a wide variety of control proteins-in this sense such proteins have calmodulinlike properties.…”

Repeated evolution of tricellular (and bicellular) pollen

“…However, Eady et al (1994) reported that both tricellular pollen of Arabidop.9is and bicellular pollen of Nicotiana took 8 d to progress from the microspore stage to anther opening under greenhouse conditions. Since both have dehydrated pollen (Johnson and McCormick, 2001;Hrubá et al, 2005), development of tricellular pollen in Arabidopsis appears to be accelerated relative to bicellular pollen in Nicotiana (Fig. 5).…”