Identification And Functional Analyses Of A Nelumbo Nucifera Annexin Involved In Seed Thermotolerance And Vigor

High temperature is one of the major environmental factors that negatively impact plant growth and productivity. As sessile organisms, plants are more vulnerable to temperature fluctuations. To cope with these challenges, plants have evolved a variety of adaptive mechanisms. Annexins are multifunctional proteins characterized by their capacity to bind calcium ions and negatively charged lipids. There is increasing evidence showed that plant annexins may acting as peroxidases and implicating their importance in plant stress responses. However, the involvement of plant annexins in high temperature stress response has not yet been described.Although molecular aspects of high temperature tolerance in plants have been investigated, few studies have been conducted in seeds in spite of their importance as a source of valuable genetic materials. Sacred lotus (Nelumbo nucifera Gaertn.) seed appeared to have the ability to withstand extremely high temperatures. Therefore, it could provide an excellent system for discovering new components involved in seed’s adaptive mechanisms. In this study, comparative proteomic analysis was used to identify novel components in heat stress signaling and leading to the identification of a sacred lotus annexin NnANN1. The functions of NnANN1 in high temperature stress and in seed vigor were also assessed. The main results obtained in this study were as follows:1. To demonstrate basal thermotolerance of sacred lotus seeds, mature sacred lotus seeds were treated at high temperatures for 24 h. Approximately, 50% of sacred lotus seeds remained alive and geminated after subjected to 90°C treatment for 24 h. Proteins were extracted from embryo axes of sacred lotus seeds that were treated at 90°C for 24 h, and untreated embryos were used as control. To identify proteins responsive to high temperature in sacred lotus seeds, both treated and untreated protein samples were resolved by 2D gel electrophoresis for comparative proteomic analysis. Ten protein spots displaying more than two fold up-regulation in response to heat stress were identified and most of the proteins are energy metabolism-related or stress-related proteins. Among the heat-responsive proteins, protein spot 2 which was identified as an annexin was chosen for further study.2. The results of quantitative Real-time PCR (qRT-PCR) and western blot showed that compared to untreated seeds, the expression of NnANN1 increased considerably in both mRNA and protein level in high-temperature treated seeds, demonstrated that NnANN1 is heat responsive, concurring with the observations obtained from our proteomic analysis. Subcellular localization analysis of NnANN1 showed that NnANN1 exhibited a typical cytosolic localization, and qRT-PCR analysis revealed that NnANN1 is expressed preferentially during seed development and germination, suggesting some fundamental function of this protein in seed development and germination.3. In an attempt to prove the in vivo protective function for NnANN1, we studied its functions in both E.coli cells and plant seeds. Under heat stress, the NnANN1 transformed E. coli cells and Arabidopsis seeds showed increased tolerance to high temperature compared with control. Furthermore, as revealed by accelerated aging (AA), the expression of NnANN1 improved seed vigor in transgenic Arabidopsis. In contrast, seeds from T-DNA insertion mutants of the Arabidopsis paralogs AtANN1 and AtANN2 were more sensitive to heat stress and AA treatment. NnANN1 transgenic seeds showed enhanced peroxidase activities, accompanied with reduced ROS release level, which may help to explain its protective function in vivo. The results support the role of NnANN1 in ROS detoxification and indicate that NnANN1 plays an important role in thermotolerance and seed vigor. The identification of NnANN1 as a high temperature responsive protein with demonstrable effects on tolerance to high-temperature stress and AA treatment may present new possibilities for exploring molecular mechanisms of high-temperature response in plants and improving thermotolerance and seed vigor in crop plants.