Cloning And Functional Characterization Of Permease NtPIC1 Gene In Tobacco Chloroplast

Iron is an essential element for plant growth and development, playing important roles in a variety of cellular activities including cell respiration, chlorophyll biosynthesis, and DNA synthesis. In chloroplasts, iron is essential for photosynthetic electron transport and functions as a cofactor for superoxide dismutases. Thus, iron homeostasis is critical for chloroplast and plant development.To better understand the mechanisms by which iron is transported into chloroplasts, we cloned and characterized the Nicotiana tabacum homolog of AtPIC1, which is a chloroplast iron transporter in Arabidopsis thaliana. We used the molecules technology of RNA interference(RNAi) and over-expressed, respectively, transformed the NtPIC1 gene to wild-type(WT) tobacco, further defined the the role of NtPIC1 in tobacco, which was provides a richer theory research for strategy I plant to understand the mechanisms of iron metabolism of tobacco. Furthermore, we further analyzed the NtPIC1 responses to the other heavy metals, especially for NtPIC1-OX transgenic tobacco can enhance the tolerance of iron deficeny which was caused by heavy metal Cd stress, the main results are as follows:1. The cDNA of Nicotiana tabacum cv. SR-1 Nt PIC1 gene was cloned by RT-PCR. The encoded protein was localized to the chloroplast membrane and Southern blotting indicated that there is a single copy of NtPIC1 in the tobacco genome. NtPIC1 was expressed in many tissues(roots, stems, leaves, flowers) of the tobacco plant. Moreover, yeast complementation assays suggested that NtPIC1 transports iron.2. The plant expression vector pRNAi-NtPIC1 and the over-expressed plant expression vector pBI121-NtPIC1 were constructed. The NtPIC1 was transformed into the nuclear genome of wild-type tobacco by Agrobacterium mediated method. The transgenic plants(NtPIC1-RNAi plants and NtPIC1-OX plants) were identified by PCR, qRT-PCR and Southern blot.3. The chlorophyll content and iron content in chloroplast of NtPIC1-RNAi and NtPIC1-OX plants showed: NtPIC1-RNAi plants exhibit albinism, dwarfism, iron-deficient chloroplasts, and chloroplast that exhibited ultrastructural defects. NtPIC1 overexpression resulted in deep-green leaves, elevated levels of chlorophyll, more densely packed chloroplasts, and the accumulation of iron and starch grains within chloroplasts.4. The expression levels of iron metabolism related genes in NtPIC1-RNAi and NtPIC1-OX plants were analysed by qRT-PCR. NtPIC1 influenced the expression of genes involving in iron transport, iron storage, iron oxidative stress, and the biogenesis of Fe-S proteins. These results suggest that NtPIC1 influence the iron homeostasis of plant.5. We transformed Saccharomyces cerevisiae BY4741 cells expressing the empty vector pYES and pYES-NtPIC1, respectively. Through the transformation of the yeast growth in culture medium containing different metal, found that with the increase of yeast dilute concentration, compared with empty vector pYES, pYES-NtPIC1 yeast in medium contain metal Ni, Cu, Co grew weaker, which was more sensitive to these metals, and in culture medium containing heavy metal Cd, NtPIC1 transformed yeast grew better, showing the heterologous expression NtPIC1 is more tolerant for heavy metal Cd.6. Tobacco plants that overexpress NtPIC1-OX have longer roots and higher fresh weights compared to WT plants in the presence of Cd. Under Cd stress, WT plants display more chlorosis and inhibition of growth, and have lower chlorophyll concentrations compared to NtPIC1-OX plants. Importantly, NtPIC1-OX plants had higher Fe concentrations in their shoots and lower Fe concentrations in their roots compared to WT plants, with or without Cd treatment. This finding indicates that there was a higher rate of Fe transport from root to shoot in NtPIC1-OX plants. Cd concentrations in NtPIC1-OX plants were significantly lower in roots and shoots compared to those in WT plants.7. Expression of Fe transport related genes(NtPIC1 in shoots, NtNRAMP1 and NtFER1 in roots) were significantly up-regulated in NtPIC1-OX plants under Cd stress. These results suggest that shoot Fe levels were maintained by up-regulating genes involving in Fe uptake/transport. The iron-deficiency-related genes NtFRO1, NtIRT1, and NtZIP1, which have been reported to mediate Cd uptake, were down-regulated in roots of NtPIC1-OX plants. This suggests that down-regulation of these genes is responsible for the reduction in Cd uptake/accumulation in NtPIC1 transformants.8. We also found that the activities of the antioxidative enzymes SOD, POD and CAT were significantly higher in NtPIC1-OX plants than WT plants after exposure to Cd stress. Our results demonstrate that overexpression of NtPIC1 is an efficient way to increase Fe content in shoots and improve the ability of resistance to oxidative damage under Cd stress.