Overview of the work:

The authors present a three-part study where they first developed a method to extract SPP from pollen grains, then measured the ice nucleating ability of SSP and its washing water, and finally, quantified the protein content of the INMs from the washing water. The authors investigated the ice nucleating ability of different extraction steps of the pollen grain, and found that the SPP themselves were not ice active. However, the authors determined that the washing of the SPP resulted in the isolation of ice nucleating macromolecules. This procedure is well explained and well-illustrated and the investigated work is clearly described. Furthermore, the authors used two methods, fluorescence spectroscopy and quantitative protein analysis assay, to determine the protein content of the ice nucleating macromolecules. This last chemical step is of particular value to the research field of biological ice nuclei. The work presented merits publication in Biogeosciences after minor revisions; my suggested revisions are focused on additional experimental details as well as on adding important literature context and comparisons to the results and discussion sections.

General feedback:

1. Additional details on the replicates and on the uncertainty of the ice nucleation results are necessary. Can the authors comment on the number of replicates necessary to generate Figures 4. What are the uncertainties associated with the immersion freezing technique and can the authors add appropriate error bars to their freezing data in Figure 4?

2. The work is well presented but lacks discussions on the comparison of the results with previously published work in the results sections and conclusion. For example, do the results presented corroborate (or not) the work by (Dreischmeier et al., 2017)? In the results section, can the authors elaborate on the comparison of their findings with work on fungal spores (Haga et al., 2014, 2013; Kunert et al., 2019)? Can the authors discuss their findings in terms of structural studies of INMs such as (Ling et al., 2018; Šantl-Temkiv et al., 2015)? The authors mention how the intine is composed of cellulose, yet there is interesting literature in ice nucleation on the relevance of cellulose which can be mentioned (see (Hiranuma et al., 2015, 2018)). Is there any connection with SPP and lignin (see (Bogler and Borduas-Dedekind, 2020))? In an atmospheric implication section, can the authors discuss if airborne ice nuclei of unknown origin could be attributed to SPPs (like in (Lloyd et al., 2020))? Are the INMs in this work forming nanogels (see (Xi et al., 2021))? In the opinion of the authors, is the field converging towards proteinaceous material is the most important INMs? If not, why? If so, why? Discussing these details will place the presented work in the greater context of the current literature on INMs and will allow future work to more effectively build upon the work presented.

Specific comments:

Title: I’m wondering if a title highlighting all three parts of the study (SPP extraction and INM isolation) may be more representative of the work. In addition, the major finding of the work is the chemical identification of proteinaceous material, which should also be mentioned in the title. Would the authors consider revising their title along the lines of “Isolation of subpollen particles (SPP) and their ice nucleating ability: SPP are carriers of proteinaceous ice nucleating macromolecules”?

Abstract:

Are the authors interested in citing the thunderstorm asthma literature to *motivate* their research? As it currently stands, the mention of thunderstorm asthma appears to be an afterthought in the introduction. I would recommend mentioning this context already in the abstract as well as move the introduction paragraph (lines 87-99) earlier. It’s an arguably important motivation for the presented research. Recent reference (including refs therein): (Bannister et al., 2021)

Line 13: the authors state a gap in knowledge, “explanations on how these materials could distribute in the atmosphere are missing” but do not address this gap in their study. Perhaps this sentence can be reworded to address the gap indeed addressed here.

Line 16: what is meant by “loosely attached”? Van der Waals forces? Covalent bonds?

Introduction:

Lines 69-70: I would be interested to read a (Tong et al., 2015) discussion in the results section in more details.

Lines 105-109: I would suggest also placing the emphasis of this work on the identification of proteinaceous material as the ice active INMs. The authors show clear chemical evidence of these types of molecules, and this finding is interesting and important.

Methods:

Lines 116-118: add the date collection information.

Lines 145-150: I wondered whether a figure with the described shapes could be helpful for the reader to visualize this calculation.

Line 155: can the formation of the emulsion be described in more detail?

Lines 166-168: Can’t dilution have an effect on the protein structure and thus its ice nucleating ability? How did the authors control for this issue?

Linea 167-168: Why was this diameter range chosen? Can a brief explanation be given?

Results:

Lines 199-200: How does this process affect the atmospheric relevance of the results in this manuscript? Can the authors elaborate?

Line 210: “volume equivalent diameters” can be defined here.

Lines 237-240: good control experiment.

Lines 242-244: this sentence seems out of place? Move to the introduction?

Line 262: What is the mass percentage of the sample that is thought to contain these concentrations of proteins?

Figure 1: it’s not clear in the caption where images of 1a and 1b are from? Could additional details be added? Which part of the figure is copyrighted?

Figure 2: very clear! Well done!  Small addition: could the instrument be specified in the caption as well?

Figure 3: additional details in the caption of how these distributions were generated would be useful.

Figure 4: Compelling data. Good job. Related to the general feedback, a discussion of replicates and uncertainties could be reported here in the caption and displayed on the graphs.

Figure 5: Cool data!

SI: the SI for this manuscript is quite short, and I wondered whether the authors might consider including the SI into the text (easier for the reader). For example, Figure S1 could be merged within Figure 2 of the main text.

Conclusion:

Line 274: can the authors make connections between the water-soluble component to dissolved organic matter? (see (Borduas-Dedekind et al., 2019; Knackstedt et al., 2018))

Technical comments:

In many instances throughout the text, a comma is missing to separate a clause from the sentence. Ex: lines 13, 15, 18, 19, 31 and so on. (English vs German syntax and comma usage)

Line 48: “The solution is” should be “was”

References:

Bannister, T., Ebert, E. E., Williams, T., Douglas, P., Wain, A., Carroll, M., Silver, J., Newbigin, E., Lampugnani, E. R., Hughes, N., Looker, C., Mulvenna, V., Csutoros, D., Jones, P. J., Davies, J. M., Suphioglu, C., Beggs, P. J., Emmerson, K. M., Huete, A., and Nguyen, H.: A Pilot Forecasting System for Epidemic Thunderstorm Asthma in Southeastern Australia, Bull. Am. Meteorol. Soc., 102, E399–E420, https://doi.org/10.1175/BAMS-D-19-0140.1, 2021.

Bogler, S. and Borduas-Dedekind, N.: Lignin’s ability to nucleate ice via immersion freezing and its stability towards physicochemical treatments and atmospheric processing, Atmospheric Chem. Phys., 20, 14509–14522, https://doi.org/10.5194/acp-20-14509-2020, 2020.

Borduas-Dedekind, N., Ossola, R., David, R. O., Boynton, L. S., Weichlinger, V., Kanji, Z. A., and McNeill, K.: Photomineralization mechanism changes the ability of dissolved organic matter to activate cloud droplets and to nucleate ice crystals, Atmospheric Chem. Phys., 19, 12397–12412, https://doi.org/10.5194/acp-19-12397-2019, 2019.

Dreischmeier, K., Budke, C., Wiehemeier, L., Kottke, T., and Koop, T.: Boreal pollen contain ice-nucleating as well as ice-binding “antifreeze” polysaccharides, Sci. Rep., 7, https://doi.org/10.1038/srep41890, 2017.

Haga, D. I., Iannone, R., Wheeler, M. J., Mason, R., Polishchuk, E. A., Fetch, T., Kamp, B. J. van der, McKendry, I. G., and Bertram, A. K.: Ice nucleation properties of rust and bunt fungal spores and their transport to high altitudes, where they can cause heterogeneous freezing, J. Geophys. Res. Atmospheres, 118, 7260–7272, https://doi.org/10.1002/jgrd.50556, 2013.

Haga, D. I., Burrows, S. M., Iannone, R., Wheeler, M. J., Mason, R. H., Chen, J., Polishchuk, E. A., Pöschl, U., and Bertram, A. K.: Ice nucleation by fungal spores from the classes Agaricomycetes, Ustilaginomycetes, and Eurotiomycetes, and the effect on the atmospheric transport of these spores, Atmospheric Chem. Phys., 14, 8611–8630, https://doi.org/10.5194/acp-14-8611-2014, 2014.

Hiranuma, N., Möhler, O., Yamashita, K., Tajiri, T., Saito, A., Kiselev, A., Hoffmann, N., Hoose, C., Jantsch, E., Koop, T., and Murakami, M.: Ice nucleation by cellulose and its potential contribution to ice formation in clouds, Nat. Geosci., 8, 273–277, https://doi.org/10.1038/ngeo2374, 2015.

Hiranuma, N., Adachi, K., Bell, D., Belosi, F., Beydoun, H., Bhaduri, B., Bingemer, H., Budke, C., Clemen, H.-C., Conen, F., Cory, K., Curtius, J., DeMott, P., Eppers, O., Grawe, S., Hartmann, S., Hoffmann, N., Höhler, K., Jantsch, E., Kiselev, A., Koop, T., Kulkarni, G., Mayer, A., Murakami, M., Murray, B., Nicosia, A., Petters, M., Piazza, M., Polen, M., Reicher, N., Rudich, Y., Saito, A., Santachiara, G., Schiebel, T., Schill, G., Schneider, J., Segev, L., Stopelli, E., Sullivan, R., Suski, K., Szakáll, M., Tajiri, T., Taylor, H., Tobo, Y., Weber, D., Wex, H., Whale, T., Whiteside, C., Yamashita, K., Zelenyuk, A., and Möhler, O.: A comprehensive characterization of ice nucleation by three different types of cellulose particles immersed in water: lessons learned and future research directions, Atmospheric Chem. Phys. Discuss., 1–82, https://doi.org/10.5194/acp-2018-933, 2018.

Knackstedt, K., Moffett, B. F., Hartmann, S., Wex, H., Hill, T. C. J., Glasgo, E., Reitz, L., Augustin-Bauditz, S., Beall, B., Bullerjahn, G. S., Fröhlich-Nowoisky, J., Grawe, S., Lubitz, J., Stratmann, F., and McKay, R. M.: A terrestrial origin for abundant riverine nanoscale ice-nucleating particles, Environ. Sci. Technol., https://doi.org/10.1021/acs.est.8b03881, 2018.

Kunert, A. T., Pöhlker, M. L., Tang, K., Krevert, C. S., Wieder, C., Speth, K. R., Hanson, L. E., Morris, C. E., Schmale III, D. G., Pöschl, U., and Fröhlich-Nowoisky, J.: Macromolecular fungal ice nuclei in Fusarium: effects of physical and chemical processing, Biogeosciences, 16, 4647–4659, https://doi.org/10.5194/bg-16-4647-2019, 2019.

Ling, M. L., Wex, H., Grawe, S., Jakobsson, J., Löndahl, J., Hartmann, S., Finster, K., Boesen, T., and Šantl‐Temkiv, T.: Effects of Ice Nucleation Protein Repeat Number and Oligomerization Level on Ice Nucleation Activity, J. Geophys. Res. Atmospheres, 123, 1802–1810, https://doi.org/10.1002/2017JD027307, 2018.

Lloyd, G., Choularton, T., Bower, K., Crosier, J., Gallagher, M., Flynn, M., Dorsey, J., Liu, D., Taylor, J. W., Schlenczek, O., Fugal, J., Borrmann, S., Cotton, R., Field, P., and Blyth, A.: Small ice particles at slightly supercooled temperatures in tropical maritime convection, Atmospheric Chem. Phys., 20, 3895–3904, https://doi.org/10.5194/acp-20-3895-2020, 2020.

Šantl-Temkiv, T., Sahyoun, M., Finster, K., Hartmann, S., Augustin-Bauditz, S., Stratmann, F., Wex, H., Clauss, T., Nielsen, N. W., Sørensen, J. H., Korsholm, U. S., Wick, L. Y., and Karlson, U. G.: Characterization of airborne ice-nucleation-active bacteria and bacterial fragments, Atmos. Environ., 109, 105–117, https://doi.org/10.1016/j.atmosenv.2015.02.060, 2015.

Tong, H.-J., Ouyang, B., Nikolovski, N., Lienhard, D. M., Pope, F. D., and Kalberer, M.: A new electrodynamic balance (EDB) design for low-temperature studies: application to immersion freezing of pollen extract bioaerosols, Atmospheric Meas. Tech., 8, 1183–1195, https://doi.org/10.5194/amt-8-1183-2015, 2015.

Xi, Y., Mercier, A., Kuang, C., Yun, J., Christy, A., Melo, L., Maldonado, M. T., Raymond, J. A., and Bertram, A. K.: Concentrations and properties of ice nucleating substances in exudates from Antarctic sea-ice diatoms, Environ. Sci. Process. Impacts, 23, 323–334, https://doi.org/10.1039/D0EM00398K, 2021.

General Evaluation:

This is an interesting manuscript describing experiments regarding the preparation and release of molecular solutes and small subpollen particles (SPP) from birch pollen. Some of the results, for example the characterization of the SPP are novel, well described and interesting, while some other experimental procedures and results are described only poorly. Moreover, I am questioning some of the interpretation regarding the connection between SPP and ice nucleating molecules (INM), and whether the INM are proteinaceous or not.

The data presented in the current manuscript might be quite useful and, hence, may support publication. I do have, however, major concerns regarding some of the procedures and the interpretation of the experiments and their application to the atmospheric situation, see below. Moreover, in some places the manuscript is technically deficient, e.g. in the detailed description of procedures or results, sometimes making it hard to understand what the authors actually refer to. Also, the citations and reference list need more care.

Overall, I think that major revisions are certainly required, as outlined below, before the paper may become suitable for publication in Biogeosciences.

Scientific points:

1.)  As far as I understand, cytoplasm generally contains also all kinds of soluble material such as proteins, lipids, polysaccharides, DNA, RNA and inorganic ions. Using the washing procedure in the extraction method described in Figure 2, I would assume that with more and more extraction volume in step 4, these solutes become more and more dilute in samples Cx. This is apparently the case for the INM. The authors also show a very slight decrease in CNC (meaning INM concentration) between samples A, B and C01. I am convinced one would see a very similar pattern with all other soluble materials, too, i.e. with proteins, with polysaccharides, with DNA, with ions etc. The authors of the current manuscript chose to investigate the content and concentration behavior of proteins using two methods (fluorescence and Coomassie staining), which is fine. And indeed, they found a similar concentration behavior for the proteins when comparing samples A, B, and C01. (They did not analyze whether proteins decrease in the same way as do INMs in samples Cx, though.) From the concentration correlation between INM and proteins in samples A, B, and C01, they infer that the INM may be proteins.

But what if the INMs belong to another substance class, for example DNA or polysaccharides? (The latter has been suggested in previous publications, also by the current authors.) I would assume to observe a similar correlation: The INMs are somewhat diluted between samples A, B and C01. In that case, the correlation between proteins and INMs would be fortuitous and would just indicate a small dilution of (any kind of) soluble material from sample A, to B, and C01. Or another thought experiment: if the authors had chosen to measure the concentration of DNA rather than that of proteins, wouldn’t they have observed a similar concentration trend for DNA, even if the INMs had been the proteins? That would not directly imply that the INMs are DNA, wouldn’t it? So from my standpoint, the correlation between INM concentration and protein concentration is not a proof that the INMs are proteins. This fact should be stated more clearly in the conclusions and abstract, to avoid a false interpretation by readers. The sentence in lines 280-281: ‘We highlight the possibility that the ice nucleation activity of Betula pendula pollen is linked not only to polysaccharides (Pummer et al., 2015) but also to proteinaceous INM.’ goes in the right direction, but may not be enough.

2.)  Lines 206-209: ‘However, even after 1-hour ultrasonic treatment we did not find any ruptured pollen grains nor SPP (Figure S1). We believe that the usually applied extraction method, where pollen grains are only left in water and are then filtrated, do not actually yield SPP unless very fresh pollen grains are used. In this sense our method is unique and offers the possibility to study isolated SPP and gain further insight about the location of the INM within the pollen grain.’

These sentences suggest that in all previous studies on dried commercial pollen, SPP were not present in the washing water. Is this notion correct? Please make a clear statement. If so, doesn’t this imply that the INM are NOT connected to the SPP as in previous studies INM were indeed found by simply washing the dried ‘old’ pollen. Please discuss in more detail.

3.)  Lines 253-254: ‘The signal correlates with heterogeneous ice nucleation of sample A, B and C01’. I am not sure I entirely understand what is meant by 'correlates' in this context. Simply, samples A, B, and C01 show fluorescence and they also show ice nucleation? Or the ice nucleation activity, namely CNC or T_50, correlates with the strength of the fluorescence signal? Please explain in more detail.

4.)  Lines 271-272: ‘In this study we develop an extraction method that gives access to the cytoplasmic material of pollen grains, even after the grains have lost the ability to germinate and rupture.’

While I applaud the authors for the realization and description of this extraction method for dried pollen, I am missing an analysis / a connection of the results presented here to the processes occurring in the atmosphere. The authors make a big point that the release of cytoplasmic material in fresh ('living') pollen is different from that of dried ones. How can they then make any quantitative conclusions and statements on free pollen and their release of ice nucleating material?

Moreover, in the sample preparation part (line 116: ‘Freshly harvested pollen samples were collected from birch trees at the Danube Island in Vienna.’), the authors mentioned that they also collected fresh pollen in Vienna, but I did not see any comparative analysis or measurements of fresh with dried pollen. Why is that so?  The authors could have made experiments with fresh pollen using the same SPP extraction procedure, using only steps 2-4, and then analyzing the filtrates in a similar manner. Why did they not do so?

5.)  Lines 281-285: ‘INM and SPP are both contained in the cytoplasm. The abundance of INM suggests that INM and SPP might not naturally separate in the atmosphere. SPP could act as carriers of INM…‘

I was wondering whether the authors can really exclude that the observed INMs come from the outer part of the pollen. I again emphasize the fact that dried pollen release INM (as shown in previous publications), but not SPP (according to the authors’ statements) contradicts the statement that INM and SPP are both contained in the cytoplasm. If INM come from the cytoplasm AND are released even without rupture, do we need to consider two different types of INM then? Please elaborate.

6.)  I still have not understood whether the amount of washing water given in Figure 4 (and Figure S2 in the supplement) refer to cumulative volume values or not. For example, for sample C01, 1 mL of washing water was used, and hence sample C01 has a total volume of 1 mL. What about sample C02? Was another 1 mL of washing water used (cumulatively the second mL) and the total volume is again 1 mL? Or were 2 mL of water used for sample C02, giving a total volume of 2 mL, and making it cumulatively 2-3 mL of waters used. Similarly, is sample C70 10 mL in total volume with the cumulative 60-70 mL of washing water used (there is a sample C60 given in the supplement)? Please explain more clearly.

Along the same lines, I am not sure how the dilution factor in equation 1 was applied to the different sample Cx solutions, and also to the samples A, B, and D. If you use different water volumes for extraction/preparing samples A, B, Cx, and D, shouldn’t the CNC concentration be quite different? Or was that volume taken into account in the dilution factor? If yes, which solution is the reference? Solution C01? 

Minor and technical points:

7.)  Apparently, the citations and references have not been assembled very carefully and need to be revised. Here are some examples:

Line 34:  ‘Mikhail Sofiev, 2013’ This is an incorrect citation (given name should be removed), probably due to the fact that the author list is incorrect, too, see below.

Lines 43-44: ‘on a global scale (Corinna Hoose et al., 2010; C. Hoose et al., 2010).’ Apparently, these are two different references. Please indicate them correctly and use correct citations, e.g. Hoose et al. 2010a; Hoose et al. 2010b.

Line 365: The reference to Gute & Abbatt  is incomplete.

Lines 367-368: The reference to Gute et al is incomplete.

Lines 403-405: The author list is corrupted. The correct author list is: Mikhail Sofiev, Jordina Belmonte, Regula Gehrig, Rebeca Izquierdo, Matt Smith, Åslög Dahl, and Pilvi Siljamo

Some references are missing their doi.

8.)  Lines 48-49: ‘The solution is then decanted and filtrated yielding what is called pollen washing water. The washing water is shown to induce ice formation at similar temperatures as the entire pollen grains.’ The tense should be past, not present.

9.)  Lines 90-91: ‘For example, birch pollen grains were shown to germinate on leaves after light rain and release starch granules.’ This sentence needs a reference.

10.)  Lines 155-56: Please provide more information on the emulsion preparation, i.e. the type of paraffin and the concentration (ratio) of the lanolin.

11.)  Lines 158-159: I could not find the number of droplets (typically) analyzed for each sample, A, B, Cx. Please provide this information.

12.)  Lines 233-234 and Table 1: ‘only after 70 mL of washing the ice nucleation activity is entirely lost’  There are some data points at temperatures higher than -34°C, both in Figure 4 and Figure S2. Were they ignored in this statement? Also, in Table 1, the CNC values for sample D at -25°C and -34°C are given as zero. Again, I am surprised, because the n_frozen/n_total ratio in Figure 4 and Figure S2 shows values slightly larger than 0. Please elaborate.

13.) Figure 1: It is not clear to me whether the images shown in panels a ) and b) and the sketch in panel c) are original to the current work, or whether they have been taken from the given references. I do not understand what is meant in the caption by ‘information for the drawing is taken from....’