Abstract
Citrate synthase (CS) and NADP-dependent isocitrate dehydrogenase (NADP-ICDH) have been considered as candidate enzymes to provide carbon skeletons for nitrogen assimilation, i.e., production of 2-oxoglutarate required by the glutamine synthetase/glutamate synthase cycle. The CS and NADP-ICDH cDNAs were encoded for polypeptides of 402 and 480 amino acids with an estimated molecular weight of 53.01 and 45 kDa and an isoelectric point of 9.08 and 5.98, respectively. Phylogenetic analysis of these proteins in wheat across kingdoms confirmed the close relationship with Aegilops tauschii and Hordeum vulgare. Further, their amino acid sequences were demonstrated to have some conserved motifs such as Mg2+ or Mn2 binding site, catalytic sites, NADP binding sites, and active sites. In-silico-identified genomic sequences for the three homeologues A, B, and Dof CS and NADP-ICDH were found to be located on long arm of chromosomes 5 and 3, and sequence analysis also revealed that the three homeologues consisted of 13 and 15 exons, respectively. The total expression analysis indicated that both genes are ubiquitously expressed in shoot and root tissues under chronic as well as transient nitrogen stress. However, they are differentially and contrastingly expressed but almost in a coordinated manner in both the tissues. Under chronic as well as transient stress, both the genes in shoot tissue showed downregulation, lowest at 6 h of transient stress. However, in the root tissue, trend was found opposite except with exceptions. Moreover, all the three homeologues of both the genes were transcribed differentially, and the ratio of the individual homeologues transcripts to total homeologues transcripts also varied with the tissue, i.e., shoots or roots, as well as with nitrogen stress treatments. Thus, cDNA as well as genomic sequence information, apparent expression at different time point of nitrogen stress, and coordination between these enzymes would be ultimately linked to nitrate assimilation and nitrogen use efficiency in wheat.
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Zhang, N., Gibon, Y., Gur, A., Chen, C., Lepak, N., Hohne, M., Zhang, Z., Kroon, D., Tschoep, H., Stitt, M., & Fine, E. B. (2010). Quantitative trait loci mapping of carbon and nitrogen metabolism enzyme activities and seedling biomass in the maize IBM mapping population. Plant Physiology, 154(4), 1753–1765.
Nunes-Nesi, A., Fernie, A. R., & Stitt, M. (2010). Metabolic and signaling aspects underpinning the regulation of plant carbon nitrogen interactions. Molecular Plant, 3(6), 973–996.
Stitt, M., Lunn, J., & Usadel, B. (2010). Arabidopsis and primary photosynthetic metabolism: more than the icing on the cake. Plant Journal, 61(6), 1067–1091.
Sienkiewicz-Porzucek, A., Nunes-Nesi, A., Sulpice, R., Lisec, J., Centeno, D. C., Carillo, P., Leisse, A., Urbanczyk-Wochniak, E., & Fernie, A. R. (2008). Mild reductions in mitochondrial citrate synthase activity result in a compromised nitrate assimilation and reduced leaf pigmentation but have no effect on photosynthetic performance or growth. Plant Physiology, 147(1), 115–127.
Scheible, W. R., Krapp, A., & Stitt, M. (2000). Reciprocal diurnal changes of phosphoenolpyruvate carboxylase expression and cytosolic pyruvate kinase, citrate synthase and NADP-isocitrate dehydrogenase expression regulate organic acid metabolism during nitrate assimilation in tobacco leaves. Plant, Cell and Environment, 23(11), 1155–1167.
Kraiser, T., Gras, D. E., Gutierrez, A. G., Gonzlez, B., & Gutierrez, R. A. (2011). A holistic view of nitrogen acquisition in plants. Journal of Experimental Botany, 62(4), 1455–1466.
Masclaux-Daubresse, C., Daniel-Vedel, F., Dechorgnat, J., Chardon, F., & Gaufichon, L. (2010). Nitrogen uptake, assimilation and remobilization in plants: challenges for sustainable and productive agriculture. Annals of Botany, 105(7), 1141–1157.
Yanagisawa, S., Akiyama, A., Kisaka, H., Uchimiya, H., & Miwa, T. (2004). Metabolic engineering with Dof1 transcription factor in plants: improved nitrogen assimilation and growth under low-nitrogen conditions. PNAS, 101(20), 7833–7838.
Ireland, R.J. and Lea, P.J. (1999). The enzymes of glutamine, glutamate, asparagine and aspartate metabolism. In BK Singh, ed, Plant amino acids: biochemistry and biotechnology. Marcel Dekker, New York, pp. 49–109.
Galvez, S., Roche, O., Bismuth, E., Brown, S., Gadal, P. and Hodges, M. (1998). Mitochondrial localization of a NADP-dependent isocitrate dehydrogenase isoenzyme by using the green fluorescent protein as a marker. Proceedings of the National Academy of Sciences, USA, 95, 7813–7818.
Sinha, S. K., Rani, M., Bansal, N., Gayatri, V. K., & andMandal, P. K. (2015). Nitrate starvation induced changes in root system architecture, carbon:nitrogen metabolism, and miRNA expression in nitrogen-responsive wheat genotypes. Applied Biochemistry and Biotechnology, 6, 1299–1312.
Gasteiger, E., Hoogland, C., Gattiker, A., Duvaud, S., Wilkins, M. R., Appel, R. D., et al. (2005). Protein identification and analysis tools on the ExPASy server. In The proteomics protocols handbook (pp. 571–607). New York: Humana.
Petersen, T. N., Brunak, S., Heijne, G. V., & Nielsen, H. (2011). SignalP 4.0: discriminating signal peptides from transmembrane regions. Nature Methods, 8(10), 785–786.
Gomi, M., Akazawa, F., & Mitaku, S. (2000). SOSUI signal: software system for prediction of signal and membrane protein. Genome Information, 11, 414–415.
Solovyev, V.V. (2007) Statistical approaches in Eukaryotic gene prediction. In Handbook of statistical genetics, Wiley-Interscience; 3d edition, 1616.
Tamura, K., Stecher, G., Peterson, D., Filipski, A., & Kumar, S. (2013). MEGA6: molecular evolutionary genetics analysis version 6.0. Molecular Biology and Evolution, 30(12), 2725–2729.
Schmittgen, T. D., & Livak, K. J. (2008). Analyzing real-time PCR data by the comparative CT method. Nature Protcols, 3(6), 1101–1108.
Foyer, C. H., Noctor, G., & Hodges, M. (2011). Respiration and nitrogen assimilation: targeting mitochondria-associated metabolism as a means to enhance nitrogen use efficiency. Journal of Experimental Botany, 62(4), 1467–1482.
Lancien, M., Ferrario-Mery, S., Roux, Y., Bismuth, E., Masclaux, C., Hirel, B., Gadal, P., & Hodges, M. (1999). Simultaneous expression of NAD-dependent isocitrate dehydrogenase and other Krebs cycle genes after nitrate resupply to short-term nitrogen-starved tobacco. Plant Physiology, 120(3), 717–725.
Agren, G. I., & Ingestad, T. (1987). Root:shoot ratio is a balance between nitrogen productivity and photosynthesis. Plant, Cell & Environment, 10, 579–586.
Cooper, H. D., & Clarkson, D. T. (1989). Cycling of amino-nitrogen and other nutrients between shoots and roots in cereals: a possible mechanism integrating shoot and root in the regulation of nutrient uptake. Journal of Experimental Botany, 40(7), 753–762.
Hu, Z. R., Han, Z. F., Song, N., Chai, L. L., Yao, Y. Y., Peng, H. R., Ni, Z. F., & Sun, Q. X. (2013). Epigenetic modification contributes to the expression divergence of three TaEXPA1 homoeologs in hexaploid wheat (Triticum aestivum). New Phytology, 197(4), 1344–1352.
Hu, Z. R., Yu, Y., Wang, R., Yao, Y. Y., Peng, H. R., Ni, Z. F., & Sun, Q. X. (2011). Expression divergence of TaMBD2 homoeologous genes encoding methyl CpG-binding domain proteins in wheat (Triticum aestivum L.). Gene, 471(1-2), 13–18.
Acknowledgments
Authors would like to acknowledge the Project Director of ICAR-NRCPB, New Delhi for his support and encouragement at various levels to execute this work. For extraction of the genomic sequence information for this work, we gratefully acknowledge IWGSC. We are also thankful to Dr. Anju M. Singh, Division of Genetics, Indian Agricultural Research Institute, New Delhi, for providing HD-2967 seeds.
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The present work was financially supported by CIMMYT under Wheat Competitive Grants fund.
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Gayatri, Rani, M., Mahato, A.K. et al. Homeologue Specific Gene Expression Analysis of Two Vital Carbon Metabolizing Enzymes—Citrate Synthase and NADP-Isocitrate Dehydrogenase—from Wheat (Triticum aestivum L.) Under Nitrogen Stress. Appl Biochem Biotechnol 188, 569–584 (2019). https://doi.org/10.1007/s12010-018-2912-2
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DOI: https://doi.org/10.1007/s12010-018-2912-2