Molecular Mechanism Analysis Of Urea Transport Of Aquaporin CsNIP2;1 In Cucumis Sativus L.

Nitrogen (N) is required for growth and development of plants, and urea is not only supplied as the major N source in agricultural fertilizers but also is an important N metabolite in plants. Currently, urea uptake into plant roots is mediated by two types of transporters:the DUR3 orthologues and the major intrinsic proteins (MIPs), mediating high-and low-affinity urea transport, respectively. It is reported DUR3 orthologues which mediate active transport of urea exist in Arabidopsis thaliana, Oryza sativa, and Zea mays and so on. However, MIPs (alias aquaporins) form a large and diverse superfamily of proteins that mediate passive flux of urea and other small nutrient solutes across biological membranes. Unlike high-affinity urea transporter, aquaporins play a significant role in transport system of small nutrient solutes besides urea. Therefore, the identification and characterization of urea transporters in higher plants is important for fundamental understanding of urea-based N-nutrition in plants and for designing novel strategies for improving the N use-efficiency of urea-based-fertilizers.Cucumis sativus L. is one of the important economic crops, and stands for cucurbitaceous dicotyledon crops. Here we characterized a MIP family member from Cucumis sativus L., CsNIP2;1, and identified it on urea transport function, transport property, subcellular localization, tissue-specific expression and expression pattern. We found that:1. Homologous sequence alignment analysis in cucumber genome indicated aquaporin CsNIP2;1 is highly similar to urea transporter CpNIPl from zucchini. Bioinformatics analysis showed CsNIP2;1 is 867bp with coding 288 amino acid, posses six transmembrane and N- and C" termi that are inside the cell, and two conserved NPA/NPA(V) located inside and outside the cell, respectively;2. The urea uptake-defective Saccharomyces cerevisiae mutant can not grow well in low concentration urea as the sole nitrogen source. Heterologously expressed CsNIP2;1 could rescue growth of the urea uptake-defective yeast on mediates with urea as the sole N source, and CsNIP2;1 permeability was inhibited by high pH. Furthermore, in the presence of different osmotically active compounds or different concentration glycerol, yeast functional complementation test showed CsNIP2;1 is permeable to urea and water but not glycerol;3. After selecting the transgenic A. thaliana expressed CsNIP2;1 promotor, GUS histochemical analysis was conducted on the seedling and mature tissues such as flower and siliques of transgenic plants, indicating CsNIP2;1 expressed highly in root, cotyledon and also expressed in calyx and siliques. Then we used qPCR analysis to test transcript abundance of CsNIP2;1 during seed germination in cucumber, and also used GUS histochemical analysis to test CsNIP2;1 promotor activity in transgenic plants during germination stage. These data suggested the expression of CsNIP2;1 was high during early seed developmental stage in plant;4. Establishing a procedure for the isolation of viable protoplasts from cucumber mesophyll cells according to sucrose density gradient centrifugation, recombination expression vector CsNIP2;1-EGFP was constructed. After isolating intact C. sativus and A. thaliana protoplasts, PEG transfection medthod was chosen for gene transient expression. These results showed CsNIP2;1-EGFP-mediated fluorescence (green) was located at the periphery of transfected protoplasts, overlapping with the lipophylic dye, FM4-64-mediated fluorescence (red), suggesting CsNIP2;1 is located at the plasma membrane in plant;5. We used qPCR analysis to test the transcript expression of CsNIP2;1 in roots of cucumber under different nitrogen sources. And we also used GUS histochemical analysis to test the activity of CsNIP2;1 promoter in transgenic plants exposing to different nitrogen sources. These data showed the expression of CsNIP2;1 is up-regulated by N deficiency, urea and nitrate;6. Wild-type A. thaliana and urea-uptake defecitve A. thaliana mutant dur3-3 were used as materials for growth phenotype analysis. The mutant atdur3-3 loses the ability for uptaking enough nitrogen and shows N deficiency phenotype. After constructing recombination expression vector CsNIP2;1-Early102, CsNIP2;1 was transformed into Wt and atdur3-3 for selecting homozygous transgenic A. thaliana expressed CsNIP2;1, respectively. Phenotype assay indicated ectopic expression of CsNIP2;1 improves growth of wild-type A. thaliana and rescues growth and development of the atdur3-3 mutant on medium with low concentration urea as a single N source, suggesting CsNIP2;1 can transport urea in plant.These data and results showed CsNIP2;1 is a plasma protein that mediate urea transport, which play an important role in urea recycle and metabolism system, including urea uptake from external environment into roots, internal urea transport during N remobilization throughout seed germination and N delivery to developing tissues.