Function Analysis Of Nelumbo Nucifera Metallothionein And Heat Shock Protein Genes In Seed Vigor

Seed vigor is an important seed quality for the success of plant propagation and food production. However, seeds gradually lose vigor and viability during storage, become more sensitive to stresses during germination and eventually lose germination ability. Although seed viability and vigor has been the subject of a number of studies, the underlying molecular mechanisms remain poorly understood.As a primitive angiosperm, sacred louts (Nelumbo nucifera Gaertn.) has been a special crop in China for nearly 5000 years. Sacred lotus seeds are characterized with 1300-year astonishing seed viability, and resistance to extreme high temperature and soilγ-radiation. Owing to these characteristics, sacred lotus seeds could therefore be an excellent plant genetic resource for identification of genes controlling seed viability and vigor.Metallothioneins (MTs) are small, cysteine-rich and metal-binding proteins which are involved in metal homeostasis and scavenging of reactive oxygen species. In plants, small heat shock proteins (sHSPs) are unusually abundant and diverse proteins involved in various abiotic stresses. Although plant MTs and sHSPs have been intensively studied, their roles in seeds remain to be clearly established. In this study, we focus on the functions of NnMT2a, NnMT2b, NnMT3 and NnHSP17.5 in seeds by gene expression analysis and transgenic approach. We have used sacred lotus seeds and Arabidopsis seeds overexpressing NnMT2a, NnMT3 and NnHSP17.5 to study their unique roles in seed germination vigor under sub-optimal conditions. The main results are as following:1. We report the isolation and characterization of three MT genes, NnMT2a, NnMT2b and NnMT3, and a cytosolic class II sHSP gene NnHSP17.5 from sacred lotus seeds.2. The results of real-time RT-PCR showed that the transcripts of NnMT2a, NnMT2b and NnMT3 were highly expressed in developing and germinating sacred lotus seeds, and were dramatically up-regulated in response to high salinity and oxidative stresses. On the other hand, NnHSP17.5 was specifically expressed in seeds under normal conditions, and was strongly up-regulated in germinating seeds upon heat and oxidative stresses.3. SDS-PAGE analysis revealed that the recombinant Trx-6His-NnMT2a, Trx-6His-NnMT3 and 6His-NnHSP17.5 were highly expressed in E. coli cells, and the fusion proteins were purified by nickel-resin affinity chromatography. After purification, the recombinant proteins were injected into rabbits to produce specific antibodies.4. Transient expression of NnMT2a-YFP, NnMT3-YFP and NnHSP17.5-YFP in protoplasts prepared from Arabidopsis suspension cultured cells was evaluated by confocal microscopy after electroporation 12-16 h. The results showed that all the tested YFP fusion proteins were localized in cytoplasm and nucleoplasm.5. In order to investigate the in vivo functions of NnMT2a, NnMT3 and NnHSP17.5, we generated transgenic Arabidopsis plants overexpressing these genes under the control of the cauliflower mosaic virus 35S promoter. RT-PCR analysis demonstrated that both NnMT2a and NnMT3 were overexpressed in dry mature seeds and green developing siliques of transgenic plants, while NnHSP17.5 was overexpressed in the dry mature seeds of transgenic plants. Similarly, immunoblot analysis of the protein extracts using specific antibodies confirmed the presence of NnMT2a, NnMT3 and NnHSP17.5 in the transgenic seeds but not in the wild-type seeds.6. The in vivo functions of NnMT2a, NnMT3 and NnHSP17.5 in seeds were evaluated by subjecting dry mature seeds of transgenic and wild-type lines to accelerated aging (AA) treatments, which was used to mimic natural aging to assess seed vigor. Transgenic Arabidopsis seeds overexpressing NnMT2a, NnMT3 or NnHSP17.5 displayed improved resistance to AA treatment, indicating their significant roles in seed germination vigor. These transgenic seeds also exhibited higher superoxide dismutase activity compared to wild-type seeds after AA treatment.7. Vital stain with tetrazolium salt can be used to demonstrate the differences in seed viability. After AA treatment, the wild-type line displayed a higher number of dead seeds than those of the transgenic lines overexpressing NnMT2a and NnMT3, as indicated by the numbers of unstained seeds.8. We also showed that NnMT2a and NnMT3 conferred improved germination ability to high salinity and oxidative stress on transgenic Arabidopsis seeds. In addition, improved basal thermotolerance was observed in the transgenic seedlings overexpressing NnHSP17.5.In summary, the biological roles of NnMT2a, NnMT3 and NnHSP17.5 in seed vigor are of significantly theoretical and practical values, which may be applicable to agricultural crops such as rice, wheat, maize and soybean for the success of germplasm conservation and crop yield.