Nitrogen Regulating the Expression and Localization of Four Glutamine Synthetase Isoforms in Wheat (Triticum aestivum L.)
Abstract
:1. Introduction
2. Results
2.1. Effects of Nitrogen on Expression of TaGS Isozymes at mRNA and Protein Level
2.2. Effects of Nitrogen on Cellular Localization of Individual TaGS Isozymes at Protein Level
2.3. Effects of Nitrogen Nutrition on GS Isozyme and Total GS Activity
2.4. Kinetic Properties of Recombinant TaGS Isozymes
2.5. Effects of Nitrogen Nutrition on C/N Metabolite Status
3. Discussion
3.1. Nitrogen Nutrition Regulates Protein Accumulation of Individual TaGS Isozymes
3.2. New Functions of TaGS Isozymes
3.3. TaGS Isozymes Synergistically Perform Nitrogen Assimilation and Translocation under Different N Nutrition Conditions
4. Materials and Methods
4.1. Plant Growth Conditions and Experimental Design
4.2. RNA Isolation and Quantitative Real-Time PCR
4.3. GS Activity Assay and Western Blotting
4.4. Immunolocalization Using Indirect Immunofluorescence Analysis
4.5. Expression of Recombinant Wheat GS Protein in E. coli
4.6. In Vitro Assay of Individual Recombinant Wheat GS Isozyme Activity
4.7. Metabolite Analysis
4.8. Statistics
Supplementary Materials
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
Abbreviations
GS | Glutamine synthetase |
N | Nitrogen |
NH4+ | Ammonium |
NO3− | Nitrate |
Gln | Glutamine |
Asn | Asparagine |
Glu | Glutamate |
Asp | Aspartate |
Ala | Alanine |
AS | Asparagine synthase |
NUE | Nitrogen use efficiency |
CDS | Coding sequence |
GMH | γ-glutamyl monohydroxamate |
IPTG | Isopropyl-b-d-thiogalactoside |
TLC | Thin layer chromatography |
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Name | Km * (mM) | Vmax ** (nKat/1 Unit Protein) | ||
---|---|---|---|---|
Glu | Hydroxylamine | Glu | Hydroxylamine | |
TaGS1;1 | 0.65 ± 0.01 d | 0.26 ± 0.02 c | 0.13 ± 0.001 d | 0.1 ± 0.002 d |
TaGS1;2 | 0.87 ± 0.01 c | 0.66 ± 0.003 a | 0.5 ± 0.002 c | 0.36 ± 0.003 c |
TaGS1;3 | 4.13 ± 0.35 a | 0.64 ± 0.04 a | 1.95 ± 0.07 a | 1.63 ± 0.08 a |
TaGS2 | 2.43 ± 0.27 b | 0.36 ± 0.04 b | 0.92 ± 0.04 b | 1.00 ± 0.04 b |
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Wei, Y.; Wang, X.; Zhang, Z.; Xiong, S.; Meng, X.; Zhang, J.; Wang, L.; Zhang, X.; Yu, M.; Ma, X. Nitrogen Regulating the Expression and Localization of Four Glutamine Synthetase Isoforms in Wheat (Triticum aestivum L.). Int. J. Mol. Sci. 2020, 21, 6299. https://doi.org/10.3390/ijms21176299
Wei Y, Wang X, Zhang Z, Xiong S, Meng X, Zhang J, Wang L, Zhang X, Yu M, Ma X. Nitrogen Regulating the Expression and Localization of Four Glutamine Synthetase Isoforms in Wheat (Triticum aestivum L.). International Journal of Molecular Sciences. 2020; 21(17):6299. https://doi.org/10.3390/ijms21176299
Chicago/Turabian StyleWei, Yihao, Xiaochun Wang, Zhiyong Zhang, Shuping Xiong, Xiaodan Meng, Jie Zhang, Lulu Wang, Xiaojiao Zhang, Meiqin Yu, and Xinming Ma. 2020. "Nitrogen Regulating the Expression and Localization of Four Glutamine Synthetase Isoforms in Wheat (Triticum aestivum L.)" International Journal of Molecular Sciences 21, no. 17: 6299. https://doi.org/10.3390/ijms21176299