the Creative Commons Attribution 4.0 License.
the Creative Commons Attribution 4.0 License.
| 31 Mar 2026
Online xylem water isotope monitoring and soil water content profiling reveal spatial root water uptake dynamics in sunflower
Abstract. Knowledge about plant water stress regulation mechanisms (e.g., plant stem capacitance) from in-situ observation is crucial for the study and modeling of plant root water uptake (RWU). We present a proof of concept and a first application of a simple method for online, minimally invasive monitoring of the water stable isotopic composition of sap xylem water of Helianthus annuus (sunflower) by inserting a sampling tube connected to a laser spectrometer in the plant stem. After careful calibration of our method, we applied it successfully to individual sunflower plants grown in soil columns. We followed the dynamics in stem water isotopic composition in response to changing light intensity and to depth-specific, isotopically labeled water pulses. We further establish that these isotopic dynamics matched changes in RWU profiles monitored simultaneously, independently, and non-destructively by the Soil Water Profiler. We finally highlight from modeling exercises the significance of the role of plant stem capacitance: water exchanges between xylem and stem non-conducting tissues were estimated to amount to about one sixth of RWU of Helianthus annuus, showing that the stem itself can be expected to be a quickly accessible reservoir of water for transpiration, very similar to what is found in trees.
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Status: final response (author comments only)
- RC1: 'Comment on egusphere-2026-1518', Anonymous Referee #1, 26 May 2026
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RC2: 'Comment on egusphere-2026-1518', Anonymous Referee #2, 27 May 2026
General comments
The authors present a novel and highly relevant methodological approach for ecohydrology and plant physiology. The authors successfully demonstrate minimally invasive monitoring of stem water isotopic composition in an herbaceous plant (sunflower) using a laser spectrometer coupled with a sampling tube. The manuscript is generally well structured and the figures are clear.
Despite these limitations, the work represents a promising step towards real‑time isotope monitoring in herbaceous plants, but the current level of replication, calibration verification, and modelling detail is insufficient for full acceptance. A revision that addresses the above points would substantially improve the manuscript.
Specific comments
- The case study relies on only two sunflower plants (plant #1 and plant #2). The observed differences between the two plants are interpreted in terms of experimental timing, but without statistical replication it is impossible to know whether these differences reflect genuine biological variability or are simply artefacts of the low n.
- The calibration was performed in hydroponic solution, yet the case study uses soil‑grown plants. The authors acknowledge that the calibration might need to be re‑determined for soil conditions, but they do not test whether the hydroponic‑derived calibration functions are valid under the soil condition.
- The method is described as “minimally invasive”, but the authors provide no comparison with other non‑destructive alternatives. The extent to which the drilling perturbs the plant is not quantified.
- The assumption that the stem water reservoir is well mixed and that the exchange fraction is constant during the day is a gross simplification. The authors can present a sensitivity analysis.
- The statement that the method “failed” when the tubing was inserted too deeply or too high above the soil is important. The authors should provide quantitative thresholds (e.g., maximum insertion depth, minimum stem diameter) that would allow other researchers to avoid these failures.
- “assuming no transport of water occurred across soil layers” (L199) this assumption is not justified. The authors can give former references or explanations on why lateral or vertical redistribution can be neglected
Technical Corrections
The reference list contains several formatting inconsistencies (e.g., missing journal names, incomplete page ranges). Check the journal guidelines for references.
Citation: https://doi.org/10.5194/egusphere-2026-1518-RC2
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- 1
This manuscript presents a proof-of-concept study for online monitoring of stem/xylem water isotopic composition in Helianthus annuus using stem vapor in-situ sampling coupled to laser spectroscopy, combined with non-destructive soil water content profiling (SWaP) to infer root water uptake (RWU) dynamics. The manuscript addresses an important topic because stable isotope methods for inferring RWU are increasingly used, while assumptions of instantaneous mixing and negligible internal water storage are often difficult to validate. Extending online isotope monitoring approaches from trees to herbaceous plants is also potentially valuable from a technical point of view.
I have several comments mainly related to uncertainties that could potentially arise from assumptions made during the soil layer-specific estimation of RWU and soil water isotopic composition (δsoil). As detailed below, these uncertainties could potentially have been better constrained by including, for example, a plant-free soil column (blank control) to quantify background soil water dynamics and/or by incorporating direct measurements of soil water isotope composition at different depths and time points throughout the experiment. The lack of such controls or direct measurements makes it difficult to evaluate the robustness of some of the key inferred quantities used in the subsequent analyses.
1. I am concerned about the authors’ use of SWaP-derived changes in soil water content to infer layer-specific RWU dynamics. It seems to me that changes in soil water content within a given layer may not necessarily reflect RWU alone, for example, in a soil column without plants, soil water content may still vary over time due to a number of factors including surface evaporation, redistribution/infiltration of water from upper to lower layers following isotope-labeling pulses added to a specific layer, etc.. Such changes could potentially be interpreted as RWU if not explicitly accounted for. In this sense a plant-free control column subjected to the same water additions and environmental conditions (which is lacking in the present study) would be helpful in constraining the uncertainty in SWaP-derived RWU profiles. I suggest that the authors either provide additional evidence that soil water redistribution and evaporation were negligible under their experimental conditions, or explicitly acknowledge this limitation. Ideally, future applications of this approach should benefit from including a blank soil column control to quantify background SWC dynamics unrelated to plant water uptake.
2. Related to the above concern, I also feel that the reported whole-column RWU rates shown in Fig. 2b appear relatively low compared with what might be expected for a sunflower plant at the flowering stage. For example, Fig. 2b suggests a peak whole-plant uptake rate (blue line) of only approximately 1.5–1.75 ml per 10 min under high light intensity (~1200 μmol m⁻² s⁻¹). Assuming a representative leaf-level transpiration rate of approximately 3 mmol H₂O m⁻² s⁻¹ for sunflower (which could potentially be even higher under such conditions, given that sunflower is a highly transpirational species), this uptake rate would correspond to only around 500 cm² total transpiring leaf area. This seems relatively small for a flowering sunflower plant, although actual leaf area was not reported.
3. Lines 199-209 describe how delta_soil water for each soil layer was estimated. According to this description, layer-specific detla_soil was calculated as a volume-weighted mixture of added labeled water and antecedent soil water. I have two concerns regarding this procedure: 1) without direct isotope measurement of soil water, how was delta_ant determined for each individual layer? Was a pre-established, depth-dependent isotope distribution model used to estimate delta_ant? or was delta_ant assumed identical among different layers (e.g., equivalent to the irrigation water isotopic composition)? 2) Eq. 4 also gives the impression that, once isotopically labeled water was added, the calculated delta_soil for each layer remained effectively constant thereafter. This means that temporal variation in delta_soil was neglected. This may need further justification. More generally I think that at least some attempts to make measurements of soil water isotope compositions at spatiotemporal scales would be beneficial than to just rely entirely on estimated values based on ideal assumptions.