Calreticulin expression and localization in relation to exchangeable Ca²⁺ during pollen development in Petunia

Pollen development in the anther in angiosperms depends on complicated cellular interactions associated with the expression of gametophytic and sporophytic genes which control fundamental processes during microsporo/gametogenesis, such as exo/endocytosis, intracellular transport, cell signaling, chromatin remodeling, and cell division. Most if not all of these cellular processes depend of local concentration of calcium ions (Ca²⁺). Work from our laboratory and others provide evidence that calreticulin (CRT), a prominent Ca²⁺-binding/buffering protein in the endoplasmic reticulum (ER) of eukaryotic cells, may be involved in pollen formation and function. Here, we show for the first time the expression pattern of the PhCRT1 gene and CRT accumulation in relation to exchangeable Ca²⁺ in Petunia hybrida developing anther, and discuss probable roles for this protein in the male gametophyte development.

Using northern hybridization, western blot analysis, fluorescent in situ hybridization (FISH), immunocytochemistry, and potassium antimonate precipitation, we report that PhCRT1 is highly expressed in the anther and localization pattern of the CRT protein correlates with loosely bound (exchangeable) Ca²⁺ during the successive stages of microsporo/gametogenesis. We confirmed a permanent presence of both CRT and exchangeable Ca²⁺ in the germ line and tapetal cells, where these factors preferentially localized to the ER which is known to be the most effective intracellular Ca²⁺ store in eukaryotic cells. In addition, our immunoblots revealed a gradual increase in CRT level from the microsporocyte stage through the meiosis and the highest CRT level at the microspore stage, when both microspores and tapetal cells show extremely high secretory activity correlated with the biogenesis of the sporoderm.

Our present data provide support for a key role of CRT in developing anther of angiosperms - regulation of Ca²⁺ homeostasis during pollen grains formation. This Ca²⁺-buffering chaperone seems to be essential for pollen development and maturation since a high rate of protein synthesis and protein folding within the ER as well as intracellular Ca²⁺ homeostasis are strictly required during the multi-step process of pollen development.