A comparative study of plant water extraction methods for isotopic analyses: Scholander-type pressure chamber vs.  cryogenic vacuum distillation

Zuecco, Giulia; Amin, Anam; Frentress, Jay; Engel, Michael; Marchina, Chiara; Anfodillo, Tommaso; Borga, Marco; Carraro, Vinicio; Scandellari, Francesca; Tagliavini, Massimo; Zanotelli, Damiano; Comiti, Francesco; Penna, Daniele

doi:https://doi.org/10.5194/hess-26-3673-2022

Articles | Volume 26, issue 13

https://doi.org/10.5194/hess-26-3673-2022

© Author(s) 2022. This work is distributed under
the Creative Commons Attribution 4.0 License.

Special issue:

Water, isotope and solute fluxes in the soil–plant–atmosphere...

https://doi.org/10.5194/hess-26-3673-2022

© Author(s) 2022. This work is distributed under
the Creative Commons Attribution 4.0 License.

Articles | Volume 26, issue 13

Research article

|

14 Jul 2022

Research article |

| 14 Jul 2022

A comparative study of plant water extraction methods for isotopic analyses: Scholander-type pressure chamber vs. cryogenic vacuum distillation

Giulia Zuecco, Anam Amin, Jay Frentress, Michael Engel, Chiara Marchina, Tommaso Anfodillo, Marco Borga, Vinicio Carraro, Francesca Scandellari, Massimo Tagliavini, Damiano Zanotelli, Francesco Comiti, and Daniele Penna

Download

Final revised paper (published on 14 Jul 2022)
Preprint (discussion started on 16 Oct 2020)

Interactive discussion

Status: closed

AC: Author comment | RC: Referee comment | SC: Short comment | EC: Editor comment

- Printer-friendly version

- Supplement

RC1: 'HESS-2020-446 Review', Anonymous Referee #1, 18 Nov 2020
- AC1: 'Reply to RC1', Giulia Zuecco, 27 Mar 2021
RC2: 'Review hess-2020-446', Anonymous Referee #2, 18 Dec 2020
- AC2: 'Reply to RC2', Giulia Zuecco, 27 Mar 2021

Peer-review completion

AR: Author's response | RR: Referee report | ED: Editor decision

ED: Reconsider after major revisions (further review by editor and referees) (11 Apr 2021) by Josie Geris

AR by Giulia Zuecco on behalf of the Authors (31 Aug 2021) Author's response Author's tracked changes Manuscript

ED: Referee Nomination & Report Request started (07 Oct 2021) by Josie Geris

RR by Anonymous Referee #2 (10 Nov 2021)

RR by Anonymous Referee #3 (19 Dec 2021)

Suggestions for revision or reasons for rejection

This paper addresses very important issue in ecohydrology and particularly for water isotope-based plant water source studies. The authors compare two methods to extract plant water: Scholander-type pressure chamber water extraction (SPC) and the most commonly used method, cryogenic vacuum distillation (CVD). I believe this study is much needed, as the community is looking for a standardized protocol in plant water source studies methods to potentially solve the generalized mismatch between soil and plant water found in many studies. However, I have major concerns about the methodology used and the current version of the paper that should be addressed before publication.
Firstly, from my point of view the reasons explaining why SPC and CVD show different isotopic composition are not profoundly discussed. Particularly, I miss two very important and current references addressing this issue that should be taken into consideration and discussed doubtlessly: (1) Barbeta et al. (2021) New Phyt, uses a novel method (special centrifuge) to separate vessel water from other water in the stem and compare these two differentiate water pools with ‘bulk’ water extracted by CVD; (2) Chen et al. (2020) PNAS compared CVD extractions to water vapour transpiration measurements (with several comments associated in PNAS and Hess). Both studies show a more enriched value of the xylem and transpired water compared to CVD (like Zuecco et al.) but associate this offset to different reasons (1) heterogeneity of water pools/domains inside the stem (2) the H exchange between organic compounds (mostly cellulose) and water during CVD water extraction. I encourage the authors to take into consideration the discussion of these papers and I believe this can be of much help to solve some of the concerns that the previous reviewers and myself have.
Additionally, I have some concerns about the methodology used:
• I don’t understand the reasons behind sampling at sunset. If the authors wanted to have the maximum hydration of the tissues (in order to get enough water for analysis) they should have conducted the sampling campaign at predawn. If they wanted to sample sap water that was transpired they should have conducted the sampling campaign at the moment of maximum transpiration (mid-morning if the conditions where very dry). I cannot understand of what conditions would be representative the sunset sampling. Maybe it was just because logistic reasons but this should be either well discussed or considered a limitation of the study. Particularly, I think about the many studies, p. ex. Pfaust et al. (2015) Tree Phys, showing daily dehydration-rehydration cycles inside the stems (with a key function of parenchyma rays in this paper). In this regard, Barbeta et al. 2021 showed also an exchange between xylem and other ‘tissue’ water in the stems. With this comment I mean that xylem water collected by SPC could be influenced by other water domains in the stem depending on the time of the day.
• In the same line as the other referees, I wonder about the contamination from phloem or other living cells during SPC sap extraction, particularly when the authors say that the extracted water was coloured (to me an indicator that there is mixing of other water in the stem apart from sap). I think no more analysis can be conducted in the samples from this study but if the authors want to promote this method this is an important issue to solve. Maybe it would be necessary not to reach too high pressures (so, the method would not be valid in very dry conditions) and analyse for organic compounds as a proxy of other tissues contamination. In this regard, in both Geißler et al. (2019) and Barbeta et al. (2021) xylem water was always less contaminated by organics than bulk (CVD). I would also suggest to the authors to make a test conducting SPC with and without leaves, bark and phloem and check differences in water isotopes and the presence of organic compounds.
• The sampling of CVD-WC or CVD-TwB, as now addressed, does not give any relevant information to the study. I would suggest to remove these results or justify better their significance.
• Do you have any soil data from these or previous campaigns? It would be good to see if soil water fitted better to SPC or CVD samples. Anyway, the overlap of SPC values to the LMWL (lower lc-excess) would indicate to me that the isotopic values for SPC are closer to the water that plants uptake and transpire than the CVD ones.
• Extraction times: Did you take note of the extraction times for each sample? It would be good to check if there is a relation between extraction time and evaporation. However, this relationship, could be also associated to a more enriched sap water inside the vessels (Martín-Gómez et al. 2016. Tree Phys)
• I agree that the fact that the isotope composition of SPC samples is not enriched like the CVD leaves, suggest that during the SPC water extraction no strong contamination from leaves water was happening. However, I would suggest the authors an additional use for the CVD-leaves isotopic composition. By building a regression line of the CVD-leaves it’s possible to retrieve the origin of the leaf evaporative water line; and check the possible water sources of the leaves (Barbeta et al. 2021).
• There is a lack of analysis and discussion about species-specific differences between the methods. For example, for beech the difference between SPC and CVD is smaller than in the other species. Could these differences be associated to wood anatomy (vessel area, wood density, parenchyma volume fraction…) or wood properties (quantity of lignin…)? Please check numerically and discuss it.

Apart from these general comments, that will necessarily change the content and structure of the paper in several parts (please include them along all the document), I have some minor comments outlined in the text:
L18. CVD-leaves is not comparable to SPC in twigs.
L21: soil water sources
L21: rephrase xylem water transpiring during the sampling day. Maybe just ‘sap water’ or ‘xylem water’
L36-37. For soils I would not say ‘a little’. Update the references for plant water extraction techniques
L45. Update the techniques (Barbeta et al. 2021, Zhao et al. 2016)
L48. Also, water within cell walls.
L48 and L52. Consider that water inside living cells might not be just different because of the age of the water but also because of fractionation inside the plant (aquaporins)
L86. Which other ecophysiological method could be used for this purpose?
L92. Any of these studies checked if the isotopic composition of SPC extracted water fitted with soil water?
L99. Urgently needed to find the best method to sample transpiration water.
L101. Consider again differences not only associated to different ages but also to internal cell fractionation. In Barbeta et al. 2021, they prove that the stem water (both xylem and other tissue water) is replaced within 3 days of well water conditions. Besides, they observe a more or less constant offset between sap and bulk water.
Table 1 and analysis. Are there species-specific differences in the quantity of water collected in the SPC and CVD twig samples?
Table 1. Did you take note of the pressure and time of the extraction? If you did, I would include the values in the table.
L150. Rephrase ‘bad injections’, change for “problems with the analyser”?
L162. Please explain “SPC contains a cutting board…” consider removing it or putting the sentence in context
L162. Consider rephrasing “The set up consisted…” one or more leaves alone? I guess you meant “one branch/twig with one or more leaves…”
L172. It would be good to have the water deficit conditions of every site (i.e. water potential of every species). Table 1?
L 174. Did you consider not to flow all the water out of the twig samples with SPC? Did you do this for any physiological reason or because you were not collecting enough water?
L 175. Wood anatomy, particularly xylem anatomy could explain also the different extraction times? For example, in Ressi, did you find differences between the two species?
L182. What does it mean fractionated in this sentence? Please rephrase or explain
L185-186. Rephrase this part also. I don’t understand the ‘use’ of CVD total plant water
L192-195. I would put this paragraph in the introduction or discussion, not in methods
L238. Evaporation + other fractionation processes
Figure 4. Leaf evaporative water line. The legend for the colours is lacking
Figure 5 and 6 are redundant to lc-excess in Figure 7. Consider to remove or move them to Supplementary Material
L293. Again, I don’t think you can compare CVD-leaves with SPC.
Discussion: you write the study limitations in two different sections. I would suggest to reorganise all the discussion and refocus your main message with the suggestions I wrote in the first part of the review.
L342-343. Explain better that statement or remove it. I think, even though CVD from twigs, trunk and branches are closer to LMWL, this is not unequivocal prove of no fractionation. If you look into the results in detail you can see that the offset for d2H is generally larger than for d18O, and not drawing an evaporative line, which could indicate a fractionation caused for a different process than evaporation (mixing, biochemical processes, etc.). Obviously, the leaves are the most fractionated/evaporated samples. Please clarify the term fractionation along the document.
L348. Please rephrase. With this sentence I could understand that all the methods could be valid. Again, clarify fractionation.
L369. Remove the sentence into () or the word “particularly”

References:
Barbeta, A., Burlett, R., Martín-Gómez, P., Fréjaville, B., Devert, N., Wingate, L., Domec, J.-C. and Ogée, J. (2021), Evidence for distinct isotopic compositions of sap and tissue water in tree stems: consequences for plant water source identification. New Phytol. https://doi.org/10.1111/nph.17857
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. Proceedings of the National Academy of Sciences, USA 117: 33345– 33350.
Martín-Gómez P, Serrano L, Ferrio JP (2017) Short-term dynamics of evaporative enrichment of xylem water in woody stems: implications for ecohydrology. Tree Physiology, 37, 511-522, http://doi.org/10.1093/treephys/tpw115
Pfautsch S, Renard J, Tjoelker MG, Salih A. 2015. Phloem as capacitor: radial transfer of water into xylem of tree stems occurs via symplastic transport in ray parenchyma. Plant Physiology 167: 963– 971.
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 & Environment 39: 1848– 1857

Hide

ED: Publish subject to revisions (further review by editor and referees) (25 Jan 2022) by Josie Geris

AR by Giulia Zuecco on behalf of the Authors (09 Mar 2022) Author's response Author's tracked changes Manuscript

ED: Referee Nomination & Report Request started (25 Mar 2022) by Josie Geris

RR by Anonymous Referee #2 (23 Apr 2022)

RR by Anonymous Referee #3 (02 May 2022)

ED: Publish subject to minor revisions (review by editor) (23 May 2022) by Josie Geris

AR by Giulia Zuecco on behalf of the Authors (07 Jun 2022) Author's response Author's tracked changes Manuscript

ED: Publish as is (26 Jun 2022) by Josie Geris

AR by Giulia Zuecco on behalf of the Authors (26 Jun 2022)

Short summary

We analyzed the variability in the isotopic composition of plant water extracted by two different methods, i.e., cryogenic vacuum distillation (CVD) and Scholander-type pressure chamber (SPC). Our results indicated that the isotopic composition of plant water extracted by CVD and SPC was significantly different. We concluded that plant water extraction by SPC is not an alternative for CVD as SPC mostly extracts the mobile plant water whereas CVD retrieves all water stored in the sampled tissue.