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Molecular identification and functional analysis of a maize (Zea mays) DUR3 homolog that transports urea with high affinity

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Abstract

Main conclusion

Successful molecular cloning and functional characterization of a high-affinity urea permease ZmDUR3 provide convincing evidence of ZmDUR3 roles in root urea acquisition and internal urea-N-remobilization of maize plants.

Urea occurs ubiquitously in both soils and plants. Being a major form of nitrogen fertilizer, large applications of urea assist cereals in approaching their genetic yield potential, but due to the low nitrogen-use efficiency of crops, this practice poses a severe threat to the environment through their hypertrophication. To date, except for paddy rice, little is known about the biological basis for urea movement in dryland crops. Here, we report the molecular and physiological characterization of a maize urea transporter, ZmDUR3. We show using gene prediction, PCR-based cloning and yeast complementation, that a functional full-length cDNA encoding a 731 amino acids-containing protein with putative 15 transmembrane α-helixes for ZmDUR3 was successfully cloned. Root-influx studies using 15N-urea demonstrated ZmDUR3 catalyzes urea transport with a K m at ~9 µM when expressed in the Arabidopsis dur3-mutant. qPCR analysis revealed that ZmDUR3 mRNA in roots was significantly upregulated by nitrogen depletion and repressed by reprovision of nitrogen after nitrogen starvation, indicating that ZmDUR3 is a nitrogen-responsive gene and relevant to plant nitrogen nutrition. Moreover, detection of higher urea levels in senescent leaves and obvious occurrence of ZmDUR3 transcripts in phloem-cells of mature/aged leaves strongly implies a role for ZmDUR3 in urea vascular loading. Significantly, expression of ZmDUR3 complemented atdur3-mutant of Arabidopsis, improving plant growth on low urea and increasing urea acquisition. As it also targets to the plasma membrane, our data suggest that ZmDUR3 functions as an active urea permease playing physiological roles in effective urea uptake and nitrogen remobilization in maize.

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Abbreviations

NUE:

Nitrogen-use efficiency

N:

Nitrogen

AN:

Ammonium nitrate

RO water:

Reverse osmosis water

FM4-64:

N-(3-Triethylammoniumpropyl)-4-(p-diethylaminophenyl-hexatrienyl) pyridinium dibromide

SD:

Synthetic dextrose

PM:

Plasma membrane

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Acknowledgments

This work was financially supported by the National Natural Science Foundation of China (NSFC) (No. 31070223, Y333ZA1D11), and the Doctoral Research Fund Project of the Ministry of Education (No. 20134320110015) as well as the Synergistic Innovation Centre for The Southern Grain and Oil Crops of China (2011-Program awarded to Hunan Agricultural University).

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Author notes

  1. Guo-Wei Liu

    Present address: Institute of Plant Biology, University of Zurich, 8008, Zurich, Switzerland

Authors and Affiliations

  1. Department of Plant Nutrition and Key Laboratory of Plant-Soil Interactions, Ministry of Education, College of Resources and Environmental Sciences, China Agricultural University, Beijing, 100193, China

    Guo-Wei Liu, Ai-Li Sun & Lai-Hua Liu

  2. Key Laboratory of Crop Physiology and Molecular Biology, National Education Ministry, Hunan Agricultural University, Changsha, Hunan, 410128, P. R. China

    Di-Qin Li & Lai-Hua Liu

  3. ARC Centre for Plant Energy Biology School of Agriculture, Food and Wine Waite Research Institute, University of Adelaide, PMB 1, Glen Osmond, SA, 5064, Australia

    Asmini Athman & Matthew Gilliham

Authors
  1. Guo-Wei Liu
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Corresponding authors

Correspondence to Di-Qin Li or Lai-Hua Liu.

Electronic supplementary material

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425_2014_2219_MOESM1_ESM.pdf

Suppl. Fig. S1 Amino acid sequence alignment of DUR3 proteins from certain plant species. Amino acids were given with standard single-letter designations, and dashes indicated gaps. Residues were shown in white letters on black if two or more sequences had identical residues at the aligned positions. The alignment was performed using Clustal-V (from DNASTARLasergene 8). DUR3 orthologs from Zea mays (Zm; KM271989), Oryza sativa (Os, AAR27948 or Os10g42960), Arabidopsis thaliana (At, At5g45380), Vitis vinifera (Vv, GSVIVG01022810001), Solanum lycopersicum (Sl, Solyc08g075570) and Populus trichocarpa (Pt, Potri.003G104000.1) (PDF 224 kb)

425_2014_2219_MOESM2_ESM.pdf

Suppl. Fig. S2 Transcriptional regulation of ZmDUR3 by nitrogen in maize roots. ZmGAPDH gene was applied as an internal reference (see materials and methods). The corresponding legend refers to the Fig. 4. Note: Samples from the NO3 −- and Gln-treated plants were not analyzed (PDF 52 kb)

425_2014_2219_MOESM3_ESM.pdf

Suppl. Fig. S3 Effect of nitrogen treatments on biomass production of Arabidopsis plants. Plants were grown for 16 days on sterile agar medium as described for Fig. 7b. The fresh weights of 30 plants in each replicate were assayed. Data are mean values ± SE (n = 3) and different letters above the bars indicate statistically significant differences (P < 0.05 by one-way ANOVA). AN, ammonium nitrate; N, nitrogen (PDF 237 kb)

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Liu, GW., Sun, AL., Li, DQ. et al. Molecular identification and functional analysis of a maize (Zea mays) DUR3 homolog that transports urea with high affinity. Planta 241, 861–874 (2015). https://doi.org/10.1007/s00425-014-2219-7

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  • DOI: https://doi.org/10.1007/s00425-014-2219-7

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