Abstract
BackgroundPollen 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.ResultsUsing 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.ConclusionOur 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.
Highlights
Pollen development in the anther in angiosperms depends on complicated cellular interactions asso‐ ciated 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
Expression pattern of the PhCRT1 gene in developing anther To analyze the expression pattern of PhCRT1 in relation to subsequent stages of pollen development, we first investigated PhCRT1 mRNA transcript levels at particular phases of microsporo/gametogenesis and in dry pollen collected from mature anther at the anther dehiscence
Expression decreased gradually through the dyad/tetrad, microspore, and pollen grain stages, and dropped significantly in dry pollen. This result is consistent with the previous studies by [13] who reported that the high levels of CRTmRNA transcripts were detected throughout anther development in Nicotiana, while CRTexpression was much lower in dry or hydrated pollen. This result indicates that the transcriptional activity of PhCRT1 or the stabilization of PhCRT1 mRNA decreases with progressive anther development and pollen maturation
Summary
Pollen development in the anther in angiosperms depends on complicated cellular interactions asso‐ ciated 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. In different plant species more genes are expressed in microspores and bicellular pollen but their number progressively declines in mature pollen [2]. There is significant overlap with the majority of genes expressed in these phases of microsporo/ gametogenesis, the percentage of specific genes increases from unicellular microspore stage to mature pollen. Progress of the microsporo/ gametogenesis depends on complicated cellular interactions associated with tissue- and cell-specific expression of gametophytic and sporophytic genes which control fundamental cellular processes during pollen formation, such as exo/endocytosis, intracellular transport, cell signaling, chromatin remodeling, and cell division. Most if not all of these processes depend of local concentration of Ca2+ [4–6]
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