Functional Study Of CML24in Regulating Pollen Germination And Pollen Tube Growth In Arabidopsis

Successful pollen germination and rapid pollen tube elongation are essential processes for sexual reproduction of flowering plants. Pollen tube elongates rapidly at its tip through the highly polarized cell growth known as tip growth, which also provides an excellent model for study of plant signaling as well as polarized tip growth.Pollen germination and tube growth are delicately regulated by external and internal signals, to maintain ion and solute homeostasis, actin dynamics, exocytosis and endocytosis, and consequent balanced pollen tube growth. Among these signals, cytosolic free Ca2+, one of the well known second messengers, plays a vital role and has been regarded as "central" player in regulating pollen germination and pollen tube growth. Therefore the investigation of Ca2+-mediated signaling pathways in pollens and pollen tubes is indispensable for better understanding of male gametophyte signal transduction for fertilization.Calmodulin-Like Proteins (CMLs) are newly discovered candidate family of Ca2+sensors in plant, with over50members in Arabidopsis thaliana. CMLs have evolutionarily conserved domain-EF-hand motif for Ca2+-binding, which is the basic feature for all Ca2+sensors and enables a conformational change after Ca2+-binding. Up to now, very little work has been done for study of CML functions in plant, not to mention in pollen tube growth for fertilization. The focus of this dissertation is to investigate the physiological roles of CML24, a member of Arabidopsis CMLs, in regulation of pollen germination and tube elongation. To achieve this, several mutant lines were obtained and used in this study, including two CML24loss-of-function mutants (one is T-DNA insertion mutant cml24-T1and another is point mutant cml24-4), CML24complementation mutants and overexpressing mutants. And the collected results are as follows:1. CML24is highly expressed in pollens and pollen tubes, and plays important roles in regulation of pollen germination and tube growth. Semi-quantitative RT-PCR and quantitative real-time PCR confirmed the expression of PTMCL1in pollens and pollen tubes, as well as the mutants used in this study are real mutants with altered transcription level of CML24. By using in vitro and in vivo pollen germination assays, we found that the germination rate, pollen tube growth rate and pollen tube length are dramatically reduced in cml24-T1and cml24-4than those of Col-0, and these phenotypes could be recovered or even enhanced in CML24complementation and overexpressing lines;2. CML24+GFP fusion protein suggest that, CML24is subcellularly expressed in cytoplasm, indicating it may serve as a cytosolic signal component and function in regulation of pollen germination and tube growth;3. cml24-T1and cml24-4mutants are hyposensitive to extracellular Ca2+and K+concentration change. Ca2+and K+are important for pollen germination and tube growth in both osmotic sustaining and signaling. They could facilitate pollen germination and tube growth at low concentration while prohibit the both processes at high concentration. The in vitro pollen germination assays by using gradient concentration of Ca2+and K+revealed that, both of the cml24-T1and cml24-4mutants are less sensitive to extracellular Ca2+and K+than Col-0;4. Fluo-3labeled Ca2+imaging show the cml24-T1and cml24-4mutants are with higher cytosolic free Ca2+than Col-0, suggests the involvement of Ca2+signal in CML24regulation of pollen germination and tube growth;5. CML24regulation of pollen germination and tube growth evokes alteration of actin organizational pattern. Actin dynamics is down regulated by [Ca2+]cyt, and the F-actin organization pattern plays a crucial role in the regulation of pollen tube growth. Alexa-488phalloidin labeled actin organization showed the disrupted actin pattern in CML24pollen tubes. In addition, the pollen germination and pollen tube growth of CML24mutants are also hyposensitive to LatB. These data indicate the importance of CML24in regulation of actin dynamics and thus to control pollen germination and tube growth;6. Preliminary data indicate that CML24answers for pollen tube orientation and NO is involved in this process. Both in vitro and in vivo pollen germination assays demonstrated abnormal pollen tube orientation in cml24-4, and this is consistent with the lower ovule fertilization rate in cml24-4. NO is a well known second messenger in the guidance of pollen tube growth. DAF-FM DA labeled cytosolic NO data showed that the mean fluorescence intensity of cml24-4pollen tube was much stronger than that of Col-0. So we may hypothesize that the alteration of NO of cml24-4mutant pollen tubes is involved in the defect of tube orientation to reach ovules to finish fertilization, and thus cause the lower seed setting rate.In summary, this study revealed that, the CML24, a member of CMLs, could positively regulate pollen germination and pollen tube growth. Loss-of-function mutants of CML24, cml24-T1and cml24-4, show decreased pollen germination rate, slower pollen tube growth and shorter pollen tube. Germinated pollens and growing tubes of cml24-T1and cml24-4mutants exhibited elevated [Ca2+]cyt levels, and also show hyposensitivity to external K+and Ca2+changes than that of wild type. Further experiments suggested that the mutant pollen tubes were with disrupted actin pattern, and less-sensitivity to LatB treatment than wild type. The cml24-4mutant pollen tubes show much higher concentration of cytosolic NO, abnormal tube orientation and lower seed setting rate. Our data suggest CML24may interact with [Ca2+]cyt, actin organization and NO, to regulate pollen germination, tube growth and orientation for successful ovule fertilization.