Isolation of subpollen particles (SPPs) of birch: SPPs are potential carriers of ice nucleating macromolecules

Abstract

Abstract. Within the last years pollen grains have gained\nincreasing attention due to their cloud-forming potential. Especially the\ndiscovery that ice nucleating macromolecules (INMs) or subpollen particles\n(SPPs) obtained from pollen grains are able to initiate freezing has stirred\nup interest in pollen. INMs and SPPs are much smaller and potentially more\nnumerous than pollen grains and could significantly affect cloud formation\nin the atmosphere. However, INMs and SPPs are not clearly distinguished. This\nhas motivated the present study, which focuses on birch pollen and\ninvestigates the relationship between pollen grains, INMs, and SPPs. According\nto the usage of the term SPP in the medical fields, we define SPPs as the\nstarch granules contained in pollen grains. We show that these insoluble SPPs\nare only obtained when fresh pollen grains are used to generate aqueous\nextracts from pollen. Due to the limited seasonal availability of fresh\npollen grains, almost all studies have been conducted with commercial pollen\ngrains. To enable the investigation of the SPPs we develop an alternative\nextraction method to generate large quantities of SPPs from commercial pollen\ngrains. We show that INMs are not bonded to SPPs (i.e. can be washed off with water). Further, we find that purified SPPs are not ice nucleation active:\nafter several times of washing SPPs with ultrapure water the ice nucleation\nactivity completely disappears. To our knowledge, this is the first study to\ninvestigate the ice nucleation activity of isolated SPPs. To study the\nchemical nature of the INMs, we use fluorescence spectroscopy. Fluorescence\nexcitation–emission maps indicate a strong signal in the protein range\n(maximum around λex\u2009=\u2009280\u2009nm and λem\u2009=\u2009330\u2009nm) with all ice nucleation active samples. In contrast, with purified SPPs\nthe protein signal is lost. We also quantify the protein concentration with\nthe Bradford assay. The protein concentration ranges from 77.4\u2009µg\u2009mL−1 (highly concentrated INMs) to below 2.5\u2009µg\u2009mL−1 (purified\nSPPs). Moreover, we investigate the connection between proteins and ice\nnucleation activity by treating the ice nucleation active samples with\nsubtilisin\xa0A and urea to unfold and digest the proteins. After this\ntreatment the ice nucleation activity clearly diminished. The results\nindicate a linkage between ice nucleation activity and protein\nconcentration. The missing piece of the puzzle could be a glycoprotein\nwhich exhibits carboxylate functionalities, can bind water in tertiary\nstructures, and displays degeneration and unfolding of its secondary\nstructure due to heat treatment or reaction with enzymes. Even though\npurified SPPs are not ice nucleation active they could act as carriers of INMs\nand distribute those in the atmosphere.\n