Are proteinaceous agglomerates responsible for ice nucleation activity of birch pollen?
Authors: Reyzek Florian , Bieber Paul , Seifried Teresa M. , Bothen Nadine , Schwidetzky Ralph , Pöschl Ulrich , Meister Konrad , Bonn Misha , Fröhlich-Nowoisky Janine , Grothe Hinrich 
Published: March 2022

&lt;p&gt;Various biological aerosol particles such as certain pollen, fungi, and bacteria are known as ice-nucleating particles with high onset freezing temperatures. It came as a surprise when Pummer et al. (2012) found that solubilized macromolecules were responsible for the ice nucleation activity of tree pollen, rather than the grains themselves. More recently, ice-nucleating macromolecules (INMs) have also been found on other tree tissues (Felgitsch et al., 2018, Seifried et al., 2020). In general, INMs are present in much greater numbers than the micrometer sized pollen grains and thus the emission of INMs from the biosphere might play a more important role than previously thought (Bieber et al., 2020, Burkart et al., 2021, Seifried et al., 2020, 2021).&lt;/p&gt;&lt;p&gt;So far, the chemical composition and structure of INMs remains largely unknown. To shine light on this, we extracted INMs from birch pollen with water and conducted various treatments, purification, and freezing experiments. For example, we detected ice nucleation activity after filtration through a 10 kDa cutoff filter. However, the concentration after 10 kDa filtration was comparatively lower than after 30 kDa or 50 kDa filtration suggesting that the INMs consist of agglomerates.&lt;/p&gt;&lt;p&gt;To concentrate the INMs we used ice affinity purification followed by treatment with acetone to precipitate proteins. We found high ice nucleation activity of this material, suggesting that the INM is an ice nucleating protein. Subsequently, size exclusion chromatography was used to isolate the INMs, leading us to a sample with high concentrations of INMs. Finally, the identification of INMs from trees will be the basis of understanding the mechanism of ice nucleation under atmospheric conditions.&lt;/p&gt;&lt;p&gt;&amp;#160;&lt;/p&gt;&lt;p&gt;References&lt;/p&gt;&lt;p&gt;Bieber, P.; Seifried, T.M.; Burkart, J.; Gratzl, J.; Kasper-Giebl, A.; Schmale, D.G., III; and Grothe, H. A Drone-Based Bioaerosol Sampling System to Monitor Ice Nucleation Particles in the Lower Atmosphere. Remote Sens., 12, 552. https://doi.org/10.3390/rs12030552, 2020.&lt;/p&gt;&lt;p&gt;Burkart, J., Gratzl, J., Seifried, T. M., Bieber, P., and Grothe, H.: Isolation of subpollen particles (SPPs) of birch: SPPs are potential carriers of ice nucleating macromolecules, Biogeosciences, 18, 5751&amp;#8211;5765, https://doi.org/10.5194/bg-18-5751-2021, 2021.&lt;/p&gt;&lt;p&gt;Felgitsch, L., Baloh, P., Burkart, J., Mayr, M., Momken, M. E., Seifried, T. M., Winkler, P., Schmale III, D. G., and Grothe, H.: Birch leaves and branches as a source of ice-nucleating macromolecules, Atmos. Chem. Phys., 18, 16063&amp;#8211;16079, https://doi.org/10.5194/acp-18-16063-2018, 2018.&lt;/p&gt;&lt;p&gt;Pummer, B. G., Bauer, H., Bernardi, J., Bleicher, S., and Grothe, H.: Suspendable macromolecules are responsible for ice nucleation activity of birch and conifer pollen, Atmos. Chem. Phys., 12, 2541&amp;#8211;2550, https://doi.org/10.5194/acp-12-2541-2012, 2012.&lt;/p&gt;&lt;p&gt;Seifried, T. M., Bieber, P., Felgitsch, L., Vlasich, J., Reyzek, F., Schmale III, D. G., and Grothe, H.: Surfaces of silver birch (Betula pendula) are sources of biological ice nuclei: in vivo and in situ investigations, Biogeosciences, 17, 5655&amp;#8211;5667, https://doi.org/10.5194/bg-17-5655-2020, 2020.&lt;/p&gt;&lt;p&gt;Seifried, T.M.; Bieber, P.; Kunert, A.T.; Schmale, D.G., III; Whitmore, K.; Fr&amp;#246;hlich-Nowoisky, J.; and Grothe, H. Ice Nucleation Activity of Alpine Bioaerosol Emitted in Vicinity of a Birch Forest. Atmosphere, 12, 779. https://doi.org/10.3390/atmos12060779, 2021.&lt;/p&gt;