Bagging: a cheaper, faster, non-destructive transpiration water sampling method for tracer studies



Stable isotope tracer experiments provide a powerful tool for understanding plant root distributions, resource uptake, niche partitioning and water cycling. Plant water is typically collected from pre-transpiring tissues to avoid the effects of evaporative isotope enrichment at the leaf surface, but extracting water from these plant samples is difficult and expensive. The purpose of this study was to test a simple transpiration bagging approach for measuring hydrologic tracer uptake.


Sampling was performed as part of a depth-specific tracer experiment in which 2H2O was injected to target depths (5, 15, 30, 60, or 150 cm) in different replicated plots. One day following injections, leaves from three species were sealed in bags for 16 h and transpired water was collected. Water from pre-transpiring stem tissue was then collected in a separate set of samples and extracted using cryogenic distillation.


Deuterium concentrations from the two techniques were correlated (R2 = 0.84) and both approaches produced similar descriptions of vertical root distributions for three dominant plant species. 18O concentrations from the two techniques were not correlated.


Bagging transpired water produced similar estimates of 2H tracer uptake as the standard sampling technique. Bagging requires no destructive sampling, specialized laboratory equipment, training or consumables and is expected to halve sampling costs. While effective in this tracer experiment, bagging may be not be effective in natural abundance experiments, or tracer experiments with very small plants or small transpiration rates (i.e., early-season or arid sites).

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  1. Asbjornsen H, Goldsmith GR, Alvarado-Barrientos MS, Rebel K, Van Osch FP, Rietkerk M, Chen J, Gotsch S, Tobon C, Geissert DR, Gomez-Tagle A (2011) Ecohydrological advances and applications in plant–water relations research: a review. J Plant Ecol 4:3–22

    Article  Google Scholar 

  2. Bakhshandeh S, Kertesz MA, Corneo PE, Dijkstra FA (2016) Dual-labeling with 15N and H218O to investigate water and N uptake of wheat under different water regimes. Plant Soil 408:429–441.

    CAS  Article  Google Scholar 

  3. Barbeta A, Ogée J and Peñuelas J (2018) Stable-isotope techniques to investigate sources of plant water. In Adv plant Ecophys tech (pp 439-456) springer, Cham

  4. Barbeta A, Gimeno TE, Clavé L, Fréjaville B, Jones SP, Delvigne C, Wingate L, Ogée J (2020) An explanation for the isotopic offset between soil and stem water in a temperate tree species. New Phytol 227:766–779.

    CAS  Article  PubMed  Google Scholar 

  5. Berry RS, Kulmatiski A (2017) A savanna response to precipitation intensity. PLoS One 12:1–18.

    CAS  Article  Google Scholar 

  6. Beyer M, Koeniger P, Gaj M, Hamutoko JT, Wanke H, Himmelsbach T (2016) A deuterium-based labeling technique for the investigation of rooting depths, water uptake dynamics and unsaturated zone water transport in semiarid environments. J Hydrol 533:627–643

    CAS  Article  Google Scholar 

  7. Beyer M, Kühnhammer K, Dubbert M (2020) In situ measurements of soil and plant water isotopes: a review of approaches, practical considerations and a vision for the future. Hydrol Earth Syst Sci 24:4413–4440

    CAS  Article  Google Scholar 

  8. Bishop K, Dambrine E (1995) Localization of tree water uptake in Scots pine and Norway spruce with hydroiogical tracers. Can J For Res 25:286–297

  9. Cernusak LA, Barbour MM, Arndt SK, Cheesman AW, English NB, Feild TS, Helliker BR, Holloway-Phillips MM, Holtum JA, Kahmen A, McInerney FA (2016) Stable isotopes in leaf water of terrestrial plants. Plant Cell Environ 39:1087–1102

    CAS  Article  Google Scholar 

  10. Chen Y, Helliker BR, Tang X, Li F, Zhou Y, Song X (2020) Stem water cryogenic extraction biases estimation in deuterium isotope composition of plant source water. P Natl Acad Sci USA 117:33345–33350

    CAS  Article  Google Scholar 

  11. Cooper LE, DeNiro MJ (1989) Covariance of oxygen and hydrogen isotopic compositions in plant water, species effects. Ecology 70:1619–1628

    Article  Google Scholar 

  12. Cui J, Tian L (2020) Temperature issues in online extraction of water from plant and soil for stable isotope analysis. Rapid Comm Mass Spec 34:8750

    Article  Google Scholar 

  13. Craig H (1961) Isotopic variations in meteoric waters. Science 133:1702–1703.

    CAS  Article  PubMed  Google Scholar 

  14. da SL Sternberg L, Bucci S, Franco A, Goldstein G, Hoffman WA, Meinzer FC, Moreira MZ, Scholz F (2005) Long range lateral root activity by neotropical savanna trees. Plant and Soil 270:169–178

  15. Dawson TE, Ehleringer JR (1993) Isotopic enrichment of water in the “woody” tissues of plants: implications for plant water source, water uptake, and other studies which use the stable isotopic composition of cellulose. Geochim Cosmochim Acta 57:3487–3492

    CAS  Article  Google Scholar 

  16. Dongmann G, Nürnberg HW, Förstel H, Wagener K (1974) On the enrichment of H218O in the leaves of transpiring plants. Rad Environm Biophys 11:41–52

    CAS  Article  Google Scholar 

  17. Dubbert M, Werner C (2019) Water fluxes mediated by vegetation: emerging isotopic insights at the soil and atmosphere interfaces. New Phytol 221:1754–1763

  18. Dubbert M, Cuntz M, Piayda A, Werner C (2014) Oxygen isotope signatures of transpired water vapor: the role of isotopic non-steady-state transpiration under natural conditions. New Phytol 203:1242–1252

    CAS  Article  Google Scholar 

  19. Ehleringer JR, Dawson TE (1992) Water uptake by plants: perspectives from stable isotope composition. Plant Cell Environ 15:1073–1082

    CAS  Article  Google Scholar 

  20. Fischer BMC, Frentress J, Manzoni S, Cousins S, Hugelius G, Greger M, Smittenberg RH, Lyon SW (2019) Mojito, anyone? An exploration of low-tech plant water extraction methods for isotopic analysis using locally-sourced materials. Front Earth Sci 7:150

    Article  Google Scholar 

  21. Grant GE, Dietrich WE (2017) The frontier beneath our feet. Water Resour Res 53:2605–2609.

    Article  Google Scholar 

  22. Holdo RM (2013) Revisiting the two-layer hypothesis: coexistence of alternative functional rooting strategies in savannas. PLoS One.

  23. Kahmen A, Schefuß E, Sachse D (2013) Leaf water deuterium enrichment shapes leaf wax n-alkane δD values of angiosperm plants I: Experimental evidence and mechanistic insights. Geochim Cosmochim Acta 111:39–49

    CAS  Article  Google Scholar 

  24. Kübert A, Paulus S, Dahlmann A, Werner C, Rothfuss Y, Orlowski N, Dubbert M (2020) Water stable isotopes in ecohydrological field research: comparison between in situ and destructive monitoring methods to determine soil water isotopic signatures. Frontier Plant Sci 11:387

    Article  Google Scholar 

  25. Kühnhammer K, Kübert A, Brüggemann N, Deseano Diaz P, van Dusschoten D, Javaux M, Merz S, Vereecken H, Dubbert M, Rothfuss Y (2020) Investigating the root plasticity response of Centaurea jacea to soil water availability changes from isotopic analysis. New Phytol 226:98–110

    Article  Google Scholar 

  26. Kulmatiski A, Adler PB and Foley KM (2020) Hydrologic niches explain species coexistence and abundance in a shrub–steppe system. J Ecol doi: 101111/1365–274513324

  27. Kulmatiski A, Adler PB, Stark JM, Tredennick AT (2017) Water and nitrogen uptake are better associated with resource availability than root biomass. Ecosphere 8:1–10.

    Article  Google Scholar 

  28. Kulmatiski A, Beard KH (2013) Root niche partitioning among grasses, saplings, and trees measured using a tracer technique. Oecologia 171:25–37.

    Article  PubMed  Google Scholar 

  29. Kulmatiski A, Beard KH, Verweij RJT, February EC (2010) A depth-controlled tracer technique measures vertical, horizontal and temporal patterns of water use by trees and grasses in a subtropical savanna. New Phytol 188:199–209.

    Article  PubMed  Google Scholar 

  30. Mamolos AP, Elisseou GK, Veresoglou DS (1995) Depth of root activity of coexisting grassland species in relation to N and P additions, measured using nonradioactive tracers. J Ecol 83:643–652

    Article  Google Scholar 

  31. Mazzacavallo MG, Kulmatiski A (2015) Modelling water uptake provides a new perspective on grass and tree coexistence. Plos One e0144300.

  32. Menchaca LB, Smith BM, Connolly J, Conrad M, Emmett B (2007) A method to determine plant water source using transpired water. Hydrol Earth Syst Sci Dis 4:863–880

    Google Scholar 

  33. Millar C, Pratt D, Schneider DJ, McDonnell JJ (2018) A comparison of extraction systems for plant water stable isotope analysis. Rapid Commun mass Spectrom 32:1031–1044.

    CAS  Article  PubMed  Google Scholar 

  34. Newberry SL, Nelson DB, Kahmen A (2017) Cryogenic vacuum artifacts do not affect plant water-uptake studies using stable isotope analysis. Ecohydrology 10:1892

    Article  Google Scholar 

  35. Ogle K, Reynolds JF (2004) Plant responses to precipitation in desert ecosystems: integrating functional types, pulses, thresholds, and delays. Oecologia 141:282–294.

    Article  PubMed  Google Scholar 

  36. Orellana F, Verma P, Loheide SP and Daly E (2012) Monitoring and modeling water-vegetation interactions in groundwater-dependent ecosystems. Rev Geophys 50(3)

  37. Ownbey GB (1991) Vascular plants of Minnesota: a checklist and atlas U of Minnesota press

  38. R Core Research Team (2004) R: a language and environment for statistical computing. R Foundation for Statistical Computing, Vienna

  39. Rothfuss Y, Javaux M (2017) Reviews and syntheses: isotopic approaches to quantify root water uptake: a review and comparison of methods. Biogeosci 14:2199–2224.

    CAS  Article  Google Scholar 

  40. Roden JS, Ehleringer JR (1999) Observations of hydrogen and oxygen isotopes in leaf water confirm the Craig-Gordon model under wide-ranging environmental conditions. Plant Phys 120:1165–1174

    CAS  Article  Google Scholar 

  41. Schymanski SJ, Sivapalan M, Roderick ML, Beringer J, Hutley LB (2008) An optimality-based model of the coupled soil moisture and root dynamics. Hydrol Earth Sys Sci 12:913–932.

    Article  Google Scholar 

  42. Silvertown J, Araya Y, Gowing D (2015) Hydrological niches in terrestrial plant communities: a review. J Ecol 103:93–108.

    Article  Google Scholar 

  43. Smithwick EAH, Lucash MS, McCormack ML, Sivandran G (2014) Improving the representation of roots in terrestrial models. Ecol Model 291:193–204.

    CAS  Article  Google Scholar 

  44. Sternberg LSL, Moreira MZ, Nepstad DC (2002) Uptake of water by lateral roots of small trees in an Amazonian tropical forest. Plant Soil 238:151–158

    CAS  Article  Google Scholar 

  45. van der Heijden G, Dambrine E, Pollier B, Zeller B, Ranger J, Legout A (2015) Mg and Ca uptake by roots in relation to depth and allocation to aboveground tissues: results from an isotopic labeling study in a beech forest on base-poor soil. Biogeochem 122:375–393.

    CAS  Article  Google Scholar 

  46. Vendramini PF, Sternberg LDS (2007) A faster plant stem-water extraction method. Rapid Comm Mass Spec 21:164–168

    CAS  Article  Google Scholar 

  47. Volkmann TH, Haberer K, Gessler A, Weiler M (2016) High-resolution isotope measurements resolve rapid ecohydrological dynamics at the soil–plant interface. New Phytol 210:839–849

    Article  Google Scholar 

  48. Walker BH, Noy-Meir I (1982) Aspects of the stability and resilience of savanna ecosystems In: Huntley BJ, Walker BH, editors Ecology of tropical savannas Berlin: Springer; 556–590

  49. Wang L, Good SP, Caylor KK, Cernusak LA (2012) Direct quantification of leaf transpiration isotopic composition. Agri Forest Meteor 154:127–135

    Article  Google Scholar 

  50. Ward D, Wiegand K, Getzin S (2013) Walter’s two-layer hypothesis revisited: Back to the roots. Oecologia 172:617–630.

    Article  PubMed  Google Scholar 

  51. Warren CP, Kulmatiski A, Beard KH (2015) A combined tracer/evapotranspiration model approach estimates plant water uptake in native and non-native shrub-steppe communities. J arid environ 121:67–78.

    Article  Google Scholar 

  52. West AG, Goldsmith GR, Brooks PD, Dawson TE (2010) Discrepancies between isotope ratio infrared spectroscopy and isotope ratio mass spectrometry for the stable isotope analysis of plant and soil waters. Rapid Comm Mass Spec 24:1948–1954

    CAS  Article  Google Scholar 

  53. Wood SN (2004) Stable and efficient multiple smoothing parameter estimation for generalized additive models. J Amer Stat Ass 99:673–686.

    Article  Google Scholar 

  54. Zhao L, Wang L, Cernusak LA, Liu X, Xiao H, Zhou M, Zhang S (2016) Significant difference in hydrogen isotope composition between xylem and tissue water in Populus euphratica. Plant Cell Environ 39:1848–1857

    CAS  Article  Google Scholar 

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Thanks to C. Carlisle, C. Pint, J. Allenbrand, and M. Holdrege for assistance with field work. K. Foley, S. Rawlings, K. Slabaugh, A. Yamaguchi, A. Zlevor, and A. Brookes assisted with sample preparation. This work was supported by grants from the US National Science Foundation Long-Term Ecological Research Program (LTER) including DEB-0620652 and DEB-1234162. Further support was provided by the Cedar Creek Ecosystem Science Reserve and the University of Minnesota. This research was also supported by the Utah Experiment Station and approved as journal paper number 9403.


Research was supported by a grant from NSF DEB, award #1354129. This research was also supported by the Utah Experiment Station and approved as journal paper number 9403.

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AK and LEF conceived of and executed the research and analyzed and wrote the manuscript.

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Correspondence to Andrew Kulmatiski.

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Kulmatiski, A., Forero, L.E. Bagging: a cheaper, faster, non-destructive transpiration water sampling method for tracer studies. Plant Soil (2021).

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  • Ecohydrology
  • Stable isotope
  • Root distribution
  • Tracer
  • Water uptake