Calmodulin activity and cAMP signalling modulate growth and apical secretion in pollen tubes

Abstract

SummaryOur present understanding implicates both calmodulin (CaM) and 3 H ,5 H -cyclicAMP (cAMP) in the regulation of pollen tube growth. However, downstream molecules of these signalling pathways and the cellular processes they modulate remain largely unknown. In order to elucidate the role of CaM, we mapped its activity in growing pollen tubes. 2-chloro-(e-amino-Lys 75 )- [6-4-(,¯uorescent analogues of CaM, were loaded into pollen tubes and CaM activity was mapped by¯uorescence ratio imaging. It was found that CaM activity exhibits a tip-focused gradient, similar to the distribution of cytosolic-free calcium ([Ca 2 ] c ). In long pollen tubes, apical CaM activity was also found to oscillate with a period similar to [Ca 2 ] c (40±80 sec). This oscillatory behaviour was not observed in small pollen tubes or in tubes that had stopped growing. Changes in CaM activity within the dome of the pollen tube apex resulting from the photolysis of caged photolysis of RS-20 (a peptide antagonist of CaM) induced re-orientation of the growth axis, suggesting that CaM is also involved in the guidance mechanism. CaM activity was strongly modulated by intracellular changes in cAMP (induced by activators and antagonists of adenylyl cyclase). These results indicate that the action of this protein is dependent not solely on [Ca 2 ] c but also on a cross-talk with other signalling pathways. A putative target of this cross-talk is the secretory machinery as observed in pollen tubes loaded with the FM (N-(3-triethylammoniumpropyl)-4-(4-dibutylamino)styryl)pyridinium dibromide 1-43 dye and exposed to different antagonists and activators of these molecules. Our data thus suggest that pollen tube growth and orientation depend on an intricate cross-talk between multiple signalling pathways in which CaM is a key element.

A loss-of-function mutation in Calmodulin2 gene affects pollen germination in Arabidopsis thaliana

“…A tipfocused gradient of cytosolic-free calcium is present in growing pollen tubes and CAM activity has been reported to display a similar tip-focused gradient (Rato et al 2004). The pivotal role played by CAM in pollen tube growth regulation has been demonstrated by the use of CAM antagonists (Rato et al 2004;Chen et al 2009) or anti-CAM antiserum (Ma et al 1999) but to our knowledge, no direct genetic evidence for a specific role for a CAM gene in pollen function was yet presented. Our data, suggesting for CAM2 a role as a Ca 2?…”

Combined Proteomic and Cytological Analysis of Ca2+-Calmodulin Regulation inPicea meyeriPollen Tube Growth

“…It has been well documented that a pivotal function of CaM that operates in both plants and animals is the regulation of cytosolic Ca 2+ level (Gilroy et al, 1987;Harper and Daly, 2000;Kaplan et al, 2006). Rato et al (2004) reported that CaM activity in the pollen tubes exhibited a tip-focused gradient, similar to the distribution of cytosol-free calcium during the polarized growth of Agapanthus umbellatus and also Lilium longiflorum, indicating that CaM may be involved in the guidance mechanism as an important Ca 2+ transducer. Previous investigations demonstrated that a rapid elevation of [Ca 2+ ] c occurred as an early event in serial self-incompatibility reactions of Papaver rhoeas pollen tubes (Geitmann et al, 2000(Geitmann et al, , 2004.…”

“…Ca 2+ -CaM plays an essential role in the interpretation of Ca 2+ signatures and their transduction to the downstream signaling components by binding to and altering the activities of various enzymes as well as cytoskeletal and structural proteins (Reddy et al, 2002;Rato et al, 2004). In gymnosperm species, the initiation of germination and the maintenance of subsequent tube elongation depended on continuous protein synthesis (Fernando et al, 2001;Hao et al, 2005), in contrast to the angiosperm pollen tube growth, in which pollen grains have presynthesized a complement of proteins (Mascarenhas, 1993;Dai et al, 2007).…”

“…Ca 2+ -CaM signaling is involved in pollen tube development (Ma et al, 1999;Rato et al, 2004), and pollen tube elongation can be inhibited by extracellular application of CaM antagonists such as trifluoperazine (TFP), calmidazolium, W-7, and W-12 (Picton and Steer, 1985;Rato et al, 2004). The CaM signaling pathways and the physiological responses that they elicit depend on the expression patterns and activities of the downstream molecules regulated by Ca 2+ -CaM.…”

Ca2+-Dependent Protein Kinase11 and 24 Modulate the Activity of the Inward Rectifying K+ Channels inArabidopsisPollen Tubes

“…The tip [Ca 2+ ] cyt oscillates at same frequency as pollen tube growth rate (Holdaway-Clarke et al, 1997;Messerli and Robinson, 1997;Messerli et al, 2000). Some Ca 2+ binding proteins, such as calmodulins, calciumdependent protein kinases (CDPKs), and cytoskeletal proteins, sense Ca 2+ signals and trigger downstream responses during pollen germination and pollen tube growth (Moutinho et al, 1998;Snowman et al, 2002;Harper et al, 2004;Rato et al, 2004;Myers et al, 2009). In particular, CDPKs are emerging as important regulators of pollen tube growth.…”