CaM is a Ca2+ sensor known to modulate the activity of many proteins. In living pollen tubes CaM seems to distribute evenly (Moutinho et al. 1998) but a higher concentration of CaM-target molecules, possibly cytoskeletal elements, was suggested to exist in the sub-apical region. The actin distribution observed in the sub-apical region of pollen tubes (Hepler et al., this volume and Yokota and Shimmen, this volume) resembles the V-shaped col lar reported for CaM binding (Moutinho et al. 1998) and thus an interaction between CaM and actin has been hypothesized. This interaction could be dependent on the levels of phosphatidylinositol (4,5)-bisphosphate (Desrivières et al. 2002), thus linking CaM to the phosphoinositide signaling pathway (Zarsky, this volume).
Although CaM distributes evenly, Rato et al. (2004) found that CaM activity is higher in the apex of growing tubes and the area of higher activity superimposes to a considerable degree with the tip-focused [Ca2+]c gradient. Furthermore, it was found that CaM activity oscillates with a period similar to [Ca] c (40-80 sec). We have also shown, as with the manipulation of [Ca + ]c in the apex (Malho and Trewavas 1996), that a decrease in CaM levels in one side of the apical dome led to growth axis reorientation to the opposite side. This clearly involves CaM in the molecular events that control pollen tube guidance (Hepler et al., this volume; Johnson and Lord, this volume). CaM might also participate in a feed-back regulation of Ca2+ stores (Sze et al., this volume). CaM can achieve regulation of Ca2+ stores and Ins(1,4,5)P3 receptors (reviewed in Malho and Camacho 2004) suggesting that CaM may allow both feedback control of membrane receptors and integration of inputs from other signaling pathways.
In addition to a role for Ca2+ in the control of CaM activation, Rato et al. (2004) provided evidence that a cAMP signalling pathway is involved. A cAMP-dependent signalling pathway in pollen was recently shown (Moutinho et al. 2001) and cAMP levels were found to be approximately uniform in the pollen tube cytosol but showing transient increases in the apical region upon reorientation and apical perturbations. CaM thus emerges as a strong candidate to integrate signals between Ca2+ and cAMP signalling pathways. Rato et al. (2004) found also that pharmacological modulation of cAMP levels caused equivalent changes in CaM activity suggesting that the activation of downstream targets of cAMP is involved in the regulation of CaM activity, possibly through [Ca2+]c.
The actin cytoskeleton and the secretory apparatus are putative candidates for a cross-regulation between signalling pathways. A growing body of evidence implicates CaM as an important receptor linking changes in Ca2+ with cytoskeletal function (Yokota and Shimmen, this volume) and Rato et al. (2004) found that a decrease in CaM levels on one side of the apical dome results in a decrease of secretory activity and reorientation. Diminishing cAMP levels mimicked this effect while an increase of cAMP (which augments CaM activity) promoted secretion. These data further support the claim for a close relationship between Ca2+ - CaM and intracellular cAMP in the control of pollen tube growth. Phosphoinositides and phospho-lipids have also been reported to modulate the actin cytoskeleton and apical secretion. Monteiro et al. (2005a, 2005b) reported that PIP2, Ins(1,4,5)P3 and phosphatidic acid (PA) regulate tip growth through a multiple pathway system involving coordinated regulation of [Ca2+]c, endo/exocytosis and vesicular trafficking. In its turn, the levels of intracellular PIP2 seem to be regulated by ROP GTPases (Kost et al. 1999; Hwang and Yang, this volume) thus linking ROP signalling to microfilament dynamics and apical secretion.
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