Distribution And Function Studies Of Calmodulin During Tobacco Pollen Germination And Pollen Tube Elongation

Calcium, a universal second messenger, act; as a mediator of stimulus-response coupling in the regulation of diverse cellular function. The changes in the cytosolic free Ca2+ could be coupled lo a changed in cell physiology and the generation of an appropriate response, which was in this case, the contraction of skeletal muscle cells. Ca2+ transducing extracellular signals within cells. It has been shown to play key roles in cell signalling in response lo a large number of extracellular signals. Ca2+ has been implicated in many apects of the eukaryotic life cycle, development, and response lo environmental cues. Ca2+ dependent modulation of cellular processes occurs via intraccllular Ca2+ binding proteins, also known as Ca2+ sensors, which decode extracellular stimuli and translate them into intracdlular signal.Calmodulin (CaM) is the most conserved calcium sensor in all eukaryotes that have been tested. CaM is an acidic EF-hand protein and it is composed of 148 amino acids arranged in two globular domains connected with a long flexible helix. Aligmenl of CaM amino acid sequences indicated a high degree of conservation between animals and plants. The ability of CaM to regulated the target enzyme activity appears to reside in hydrophobic regions that are exposed in the conformalional change that occurs upon the binding of Ca2+ lo CaM. The hallmark of CaM's mechanism is that it decodes information contained the temporal and spatial patterns of Ca2+ signals and brings about changes in metabolism and gene expression. CaM-mediated regulation may be widespread in the Ca2+ channel family. It regulates ion transporters controlling Ca2+homeostasis, which may lead to increase or decrease [Ca2+]c above a value so that pollen lube growth could be efficiently controlled The crucial role of CaM in pollen tube elongation has being been a locus in many reportsAs the male gametophyle of higher plants, pollen provides some fascinating biology. Upon landing on the stigma where they choose a germination aperture, pollen grains hydrate and germinate, producing a polarized outgrowth of pollen tube, which elongates over long distances to deliver the sperm cells to the embryo sacs within ovules for fertilization. The convenience of pollen grain storage, easy growth condition for pollen tubes in vitro, and the importance of tube growth for reproduction, have made these cells a model system for studying cellular polarity and signal transduclion. Plenty of chemical mechanism and complex signal transduclion are involved in pollen grains germination and tubes elongation. Ca2+ as a second messenger has being widely studied during pollen germination, tube elongation and orientationCaM-GFP and CaM-GUS fusion gene were constructed and were put the control of 35S promoter or LAT52-7 pollen specific promoter respectively. These constucts were transformed into Nicotiana labacwn SRI by leaf disc transformation through Agrobacterial LBA4404. Using GFP, under the con focal laser scanning system (CLSM), the spatial distribution of CaM could be delected in real time during pollen germination and pollen tube elongation in Iransgcnic tobacco, so that the cell biological study on CaM could be conducted. Our main results are given as followed:1. GUS reaction results and CaM-GFP fluorescent signal showed that CaM was widely observed in roots, stems and leaves. High levels of CaM were observed throughout the cytoplasm of root meristcm region, quiescence center cell, root hair cells, epidermal cells and vascular cells. Further more, CaM showed polar distribution under the membrane of the joint site between two guard cells or two epidermal hair cells. CaM was also observed lo localize in the cytoplasl of pollen grain with much high levels at the vegetative nucleus and the germination apertures, and tip-to-base gradient of CaM was delected in the elongating pollen tube. Our data indicated that CaM showed asymmetric distribution in plant cells, which has a cl...