Exploring Nitrogen Exudation in Cotton with HPLC and FT-ICR-MS

Drought stress could alter root exudation of nitrogen (N) in cotton (Gossypium hirsutem), according to a recent study by the Department of Soil and Crop Sciences of Texas A&M University and Texas A&M AgriLife (College Station, Texas) and the Department of Environmental Sciences of the University of Arizona (Tuscon, Arizona). This could be a mechanism of drought tolerance, similar to altered carbon (C) exudation, the scientists wrote in the study, which was published in Frontiers in Plant Sciences (1).

The composition and quantity of root exudates will respond to drought severity and recovery (2), and the response will differ by species and genotype (3-6). Understanding the effects of drought’s effects on root exudation behavior can be used to harness drought-resilient rhizosphere microbiomes for improved climate resilience in agronomic crops that are increasingly threatened by water-limited environments (7). The effect of drought and subsequent recovery on root exudation mechanisms in agriculturally relevant crops requires significant attention as well (8).

The objectives of the investigation were to quantify organic N exudation from whole root systems non-destructively and identify drought-induced shifts in the N metabolome of root exudates by comparing root exudates of drought-treated plants to well-watered control plants during progressive drought stress and subsequent recovery in a time-series experiment. Total organic C and N were quantified by combustion, inorganic N with spectrophotometric methods, free amino acids by high performance liquid chromatography (HPLC), and untargeted metabolomics by Fourier-transform ion cyclotron resonance-mass spectrometry (FT-ICR-MS [1]).

Root exudates are organic carbon compounds such as simple sugars, organic acids, and amino acids which is released from living plant roots into the soil. These small molecules can bind directly to soil minerals, which makes them important regulators of soil carbon formation and loss. Unlike plant litter (for example, leaves and roots), which must be decomposed before it can affect the soil carbon pool, root exudates can have immediate effects on mineral-associated organic matter (MAOM), which contains long-cycling, “stable” soil carbon (9) In turn, soil microorganisms, in turn, influence the plant metabolome (10), and assist plants in enduring biotic and abiotic stresses (11).

The results of the analysis indicated that organic N molecules in root exudates were by far the greatest component of root exudate total N, which accounted for 20-30% of root exudate mass. Drought increased root exudation of organic N (62%), organic C (6%), and free amino acid-N (562%), yet free amino acids were <5% of the N balance. Drought stress significantly increased root exudation of serine, aspartic acid, asparagine, glutamic acid, tryptophan, glutamine, phenylalanine, and lysine compared to the control. There was a total of 3985 molecules detected across root exudate samples, of which 41% contained N in their molecular formula. There were additionally 349 N-containing molecules unique to drought treatment and 172 unique to control. Drought increased the relative abundance and redistributed the molecular weights of low molecular weight N-containing molecules. Time-series analysis revealed root exudation of organic N was stimulated by drought and was sensitive to the degree of drought stress (1).

The paper authors state that, while the current study is limited in that results are from a soilless system, there are few alternatives to collect root exudates in time-series analysis from the same plants overtime; thus, future studies should attempt to build upon these results in soil systems. From here there is an exciting path for linking C and N exudation to microbial assembly in the rhizosphere, engineering drought resilient rhizospheres, and challenging traditional models of N biogeochemistry given the substantial allocation to root exudation of organic N during low water availability (1).

Cotton farm during harvest season. © Oleks Stock - stock.adobe.com

References

1. Coker, H. R.; Lin, H. A.; Shackelford, C. E. B.; Tfaily, M. M.; Smith, A. P.; Howe, J. A. Drought Stimulates Root Exudation of Organic Nitrogen in Cotton (Gossypium hirsutem). Front Plant Sci. 2024, 15, 1431004. DOI: 10.3389/fpls.2024.1431004

2. Lin, H.-A.; Coker, H. R.; Howe, J. A.; Tfaily, M. M.; Nagy, E. M.; Antony-Babu, S. et al. Progressive Drought Alters the Root Exudate Metabolome and Differentially Activates Metabolic Pathways in Cotton (Gossypium hirsutum). Front. Plant Sci. 2023, 14. DOI: 10.3389/fpls.2023.1244591

3. Canarini, A.; Merchant, A.; Dijkstra, F. A. Drought Effects on Helianthus annuus and Glycine max Metabolites: From Phloem to Root Exudates. Rhizosphere 2016, 2, 85–97. DOI: 10.1016/j.rhisph.2016.06.003

4. Gargallo-Garriga, A.; Preece, C.; Sardans, J.; Oravec, M.; Urban, O.; Peñuelas, J. Root Exudate Metabolomes Change Under Drought and Show Limited Capacity for Recovery. Sci. Rep. 2018, 8, 1–15. DOI: 10.1038/s41598-018-30150-0

5. Williams, A.; de Vries, F. T. Plant Root Exudation Under Drought: Implications for Ecosystem Functioning. New Phytol. 2020, 225, 1899–1905. DOI: 10.1111/nph.16223

6. Chen, Y.; Yao, Z.; Sun, Y.; Wang, E.; Tian, C.; Sun, Y. et al. Current Studies of the Effects of Drought Stress on Root Exudates and Rhizosphere Microbiomes of Crop Plant Species. Int. J. Mol. Sci. 2022, 23, 2374. DOI: 10.3390/ijms23042374

7. de Vries, F. T.; Griffiths, R. I.; Knight, C. G.; Nicolitch, O.; Williams, A. Harnessing Rhizosphere Microbiomes for Drought-Resilient Crop Poduction. Sci. (80-.) 2020, 368, 270–274. DOI: 10.1126/science.aaz5192

8. Williams, A., de Vries, F. T. Plant Root Exudation under Drought: Implications for Ecosystem Functioning. New Phytol. 2020, 225, 1899–1905. DOI: 10.1111/nph.16223

9. Researchers Discover Root Exudates Have Surprising and Counterintuitive Impact on Soil Carbon Storage. Harvard University Department of Organismic and Evolutionary Biology website. https://oeb.harvard.edu/news/researchers-discover-root-exudates-have-surprising-and-counterintuitive-impact-soil-carbon (accessed 2024-12-05)

10. Badri, D. V.; Zolla, G.; Bakker, M. G.; Manter, D. K.; Vivanco, J. M. Potential Impact of Soil Microbiomes on the Leaf Metabolome and on Herbivore Feeding Behavior. New Phytol. 2013, 198, 264–273. DOI: 10.1111/nph.12124

11. Zolla, G.; Badri, D. V.; Bakker, M. G.; Manter, D. K.; Vivanco, J. M. Soil Microbiomes Vary in Their Ability to Confer Drought Tolerance to Arabidopsis. Appl. Soil Ecol. 2013, 68, 1–9. DOI: 10.1016/j.apsoil.2013.03.007