The Study Of CML25 Regulating The Pollen Germination And Pollen Tube Growth In Arabidopsis

The successful germination of pollen grains and rapid polarized elongation of germinated pollen tubes are essential for double fertilization and consequent seed production, and Ca2+ signal is important for the both processes, but the fundamental signaling network still needs further investigation.The Ca2+ signals are perceived and transduced by various Ca2+ sensors downstream, and Calmodulin-Like Proteins (CMLs) are members of the most recently characterized Ca2+ sensor family which shares high homology with Calmodulins (CaM). CML is the biggest Ca2+ sensor family with 50 members in Arabidopsis, and their physiological roles remain largely unknown. The data from the GeneChip arrays indicated that CML25 was expressed highly in pollen grains and pollen tubes, suggested the importance of CML25 in the functioning of the male gametophyte. In this study, we investigated the physiology roles of CML25 in pollen germination and pollen tube growth. Our findings indicated that CML25 was an important positive element of [Ca2+]-regulated transmembrane K+ trafficking and consequent pollen germination and tube elongation.We firstly confirmed validated the Ca2+ -binding activity and the tissue expression of CML25 as well as its physiology roles in pollen grain germination and tube growth. By using the SDS-PAGE to verify the electrophoretic mobility shift of CML25 with/without Ca2+ incubation, the results indicated that CML25 could bind Ca2+ and make conformational change. Quantitative real-time PCR and GUS staining assay indicated that CML25 was primarily expressed in pollen grains and pollen tubes, which was consistent with the predication from the microarray. Two independent CML25 T-DNA insertion mutants, cml25-1 and cml25-2, suffered a major reduction in both the rate of pollen germination and the elongation of the tube, cml25 mutants exhibited reduced fertility. Complementation of the cml25-1 mutant resulted in the recovery of the wild type phenotype. The conclusion was that CML25 acted as a positive regulator in pollen germination and pollen tube growth.To elucidate the molecular mechanism of CML25 in regulation of pollen germination and tube growth, the subcellular localization and cytosolic free Ca2+ ([Ca2+]cyt) variation as well downstream transmembrane K+ trafficking were fully analyzed with fusion protein constructs and cml25 mutants. Sub-cellular localization assay showed that CML25-GFP was localized in the cytoplasm. Fluo-3 labeled Ca2+ imaging exhibited that the cml25 mutant pollen grains and pollen tubes harbored a higher [Ca2+]cyt than those of wild type did. The normal tip-focused Ca2+ gradient in wild type pollen tubes was altered in cml25 mutants. The changes suggested that CML25 was involved in Ca2+ signaling pathway. The cml25 mutants were less sensitive to concentration change of exogenous Ca2+ and K+. Patch-clamp whole-cell recordings demonstrated that, the inward K+ currents of mutant pollen and pollen tube protoplasts was hyposensitive to the elevated [Ca2+]cyt, suggesting that CML25 may mediate [Ca2+]cyt regulation of pollen and pollen tube K+ influx and thus to regulate pollen germination and pollen tube growth.To further explore the downstream molecular pathway of CML25, yeast two-hybrid and screening in library of Arabidopsis assays suggested that CML25 might interact with CIP1 (CML25 Interacting Protein 1), and this interaction was further confirmed with the yeast two-hybrid, bimolecular fluorescence complementation (BiFC) and the subcellular localization assays. These results showed that the interaction might happen in cytoplasm. Further pollen germination and tube growth assays by using the CIP1 overexpressing lines (CIP1-OE5, CIP1-OE6) and its transgene silence lines (CEP1-R1, CIP1-R2) indicated that CIP1 was a negative element in the regulation of pollen tube growth, but the molecular interaction mechanism between CML25 and CIP1 still needs further investigation.In conclusion, this study revealed that the pollen grain and pollen tube highly expressed CML25, was a positive cytosolic element of [Ca2+]cyt regulation of K+ transmembrane absorption during pollen germination and pollen tube growth in Arabidopsis.