Regulation of pollen tube growth by calmodulin, cyclic nucleotides and cyclic nucleotide-gated channels

Tese de doutoramento em Biologia (Biologia Celular), apresentada à Universidade de Lisboa através da Faculdade de Ciências, 2007Pollen tube growth plays an essential role in fertilization of flowering plants. Several signalling pathways were identified during pollen tube growth, including Ca2+, CaM and 3',5'-cyclic adenosine monophosphate (cAMP). These constitute a complex web of signalling networks that intersect at various levels (reviewed in Chapter I). In order to elucidate the role of CaM we mapped its activity in growing pollen tubes. We found that CaM activity exhibits a tip-focused gradient, similar to the distribution of cytosolic free Ca2+ ([Ca2+]c), and that it oscillates with a period similar to [Ca2+]c . Moreover, we show that CaM is also involved in the guidance mechanism and has its activity strongly modulated by intracellular changes in cAMP. A putative target of the crosstalk between CaM and cAMP is the secretory machinery as observed in pollen tubes loaded with the FM 1-43 dye. Our data thus suggest that pollen tube growth and orientation depends on an intricate crosstalk between multiple signalling pathways in which CaM is a key element (Chapter II). In Chapter III we study two possible targets of CaM, CNGC7 and CNGC8. These proteins are both expressed in pollen and show strong homology, suggesting functional redundancy. To gain insights into the biological function of CNGC7 and CNGC8, we used reverse-genetics. Neither CNGC7 nor CNGC8 was found to be essential, but CNGC7 appears to play a role in fertilization. To further address a possible redundancy between CNGC7 and CNGC8, we generated a double-mutant and the double knockout was found to be gametophyte lethal. These results provide the first genetic evidence for redundancy among CNGCs in fertilization. Plant CNGCs contain a C-terminal cyclic nucleotide binding domain with an overlapping CaM binding site. Cyclic nucleotides induce channel opening, while CaM mediates the feed-back inhibition of the channel. To gain insights into the structure-function relationship of plant CNGCs, we used a site-directed mutagenesis technique coupled with complementation assays of cngc18 mutants. Amino acid substitutions E520A, F565A and F565W provided partial complementation. Taken together, these results show that F565 and E520 are key amino acid residues for cyclic nucleotide binding and that changes in the cyclic nucleotide binding domain are capable of disrupting protein function (Chapter IV). In Chapter V all results obtained were discussed