A rapid and efficient method to study the function of crop plant transporters in Arabidopsis

Authors: WANG, XIANGFENG - The Chinese University Of Hong Kong (CUHK); ZHONG, FUDI - The Chinese University Of Hong Kong (CUHK); WOO, CHEUK HANG - The Chinese University Of Hong Kong (CUHK); MIAO, YANSONG - The Chinese University Of Hong Kong (CUHK); Grusak, Michael; ZHANG, XIAOBO - Zhejiang University; TU, JUMIN - Zhejiang University; WONG, YUM SHING - The Chinese University Of Hong Kong (CUHK); JIANG, LIWEN - The Chinese University Of Hong Kong (CUHK)

Submitted to: Protoplasma
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 5/13/2016
Publication Date: 5/30/2016
Publication URL: http://handle.nal.usda.gov/10113/63180
Citation: Wang, X., Zhong, F., Woo, C., Miao, Y., Grusak, M.A., Zhang, X., Tu, J., Wong, Y., Jiang, L. 2016. A rapid and efficient method to study the function of crop plant transporters in Arabidopsis. Protoplasma. doi:10.1007/s00709-016-0987-6.

Interpretive Summary: Iron deficiency in food crops results in decreased nutritional value of harvested foods. To help maintain quality food production, we are interested in understanding how iron transport proteins from different crop plants are able to move iron from the soil into plant roots, then into different plant cells, and ultimately on to edible tissues of the shoots, such as leaves or seeds. To help us evaluate the function of these iron transport proteins, we have developed a rapid and efficient method that takes advantage of mutant plants that contain defective copies of these proteins. We then take functional copies of these proteins from various crop plants and put them into the mutant plants to study how well the proteins transport iron. In this study we used an iron transporter from rice to demonstrate the effectiveness of this new method. By measuring iron and other metals in plant tissues, we showed that both isolated cells of the mutant plant, as well as fully grown plants, could be used to demonstrate the functionality of the rice iron transport protein. This method can be replicated with iron transporters from a number of important agronomic crop plants or from wild crop relatives.

Technical Abstract: Iron (Fe) is an essential micronutrient for humans. Fe deficiency disease is wide-spread and has lead to extensive studies on the mechanisms of Fe uptake and storage, especially in staple food crops such as rice. However, studies of functionally related genes in rice and other crops are often time and space demanding. Here, we demonstrate that transgenic Arabidopsis suspension culture cells and Arabidopsis plants can be used as an efficient expression system for gain-of-function study of selected transporters, using Fe transporters as a proof-of-principle. The vacuolar membrane transporters OsVIT1 and OsVIT2 have been described to be important for iron sequestration, and disruption of these two genes leads to Fe accumulation in rice seeds. In this study, we have taken advantage of the fluorescence-tagged protein GFP-OsVIT1, which functionally complements the ccc1 yeast mutant, to generate transgenic Arabidopsis suspension cell lines and plants. GFP-OsVIT1 was shown to localize on the vacuolar membrane using confocal microscopy and immunogold EM. More importantly, the Fe concentration, as well as the concentration of Zn, in the transgenic cell lines and plants were significantly increased compared to that in the WT. Taken together, our study shows that the heterologous expression of rice vacuolar membrane transporter OsVIT1 in Arabidopsis system is functional and effectively enhances iron accumulation, indicating a rapid and efficient approach for studying other putative transporters of crop plants in this system.