Subcellular Localization Of Arabidopsis Annexin Proteins And Overexpression Of ANN4 And ANN6

Annexins are Ca2+ and phospholipid binding proteins. Annexins in animal cells are involved in vesicle transport, calcium channel formation, signal transduction, cell differentiation and so on. In plant cells, Annexins participate in the secretion of plant cells, exocytosis, calcium metabolism and establishment of ion channels as well as a series of membrane activities. Plant Annexins can also regulate some complex physiological processes such as response to cold, salt, drought stresses, etc. Annexins also play important roles in scavenging reactive oxygen species and regulating the concentration of free calcium in plant cells.Previously, an Annexin gene was found to negatively regulate RPW8.1-mediated cell death and disease resistance in our Laboratory. In Arabidopsis, there are eight members of Annexin family, namely ANN1, ANN2, ANN3, ANN4, ANN5, ANN6, ANN7, and ANN8. Much work has been done on the functions of ANN1 and ANN4, whereas little is known on the roles of other members in Annexin family. Subcellular localization is considered as an important step to decipher protein functions. In this thesis, we carried out extensive analysis on the subcellular localization of eight Annexins in Arabidopsis and two in rice (OsANN and OsANNP35), using fluorescent protein reporter gene system, transient expression and transgenic approaches. The results revealed that in N. benthamiana transient expression experiments the majority of Arabidopsis Annexins (ANN1、ANN2、ANN、ANN7、ANN8) were localized in the cytoplasm, more likely binding to cell membrane, and they also formed ER (endoplasmic reticulum) rings around nuclei. ANN5-eYFP was localized in both cytoplasm and nucleus, while ANN4-eYFP and ANN6-eYFP showed unique localization patterns, i.e. ANN4-eYFP gathered as trip-like shape and ANN6-eYFP seemed to be accumulated as large spots. Taken together, the subcellular localization patterns of Arabidopsis Annexins are diverse, suggesting that they may possess a wide broad of biological functions.Moreover, we found that ANN4 and ANN6 could delay the nucleus localization of NLS-tagged RFP protein in N. benthamiana, which was contrastable to other Annexin proteins. Then, we made constructs for over-expressing ANN4 and ANN6 in transgenic Arbidopsis and further examined their subcellular localizations. Our data demonstrated that 35S::ANN4-eYFP accumulated as silk strip of aggregation and 35S::ANN6-eYFP as punctate deposition in the cytoplasm. Furthermore, transgenic Arabidopsis plants expressing 35S::ANN6-eYFP were more susceptibile to powdery mildew than wild type control, while no significant difference was detected between 35S::ANN4-eYFP and wild type (Col-gl).In summary, this study has characterized the subcellular localization patterns of Arabidopsis Annexins, and suggested some novel functions for Annexin family members, such as involvement in endocytosis, delaying NLS-targeting to nucleus, and association with disease resistance. These data provide important clues to extensively clarify the biological roles of Annexins in plant.