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Ice-nucleating proteins (INPs) catalyze ice formation at high subzero temperatures, with major biological and environmental implications. While bacterial INPs have been structurally characterized, their counterparts in other organisms remain unknown. Here, we identify a new class of efficient INPs in fungi. These proteins are membrane-free, adopt β-solenoid folds, and multimerize to form large ice-binding surfaces, showing mechanistic parallels with bacterial INPs. Structural modeling, sequence analysis, and functional assays show they are encoded by orthologs of the bacterial InaZ gene, likely acquired via horizontal gene transfer. Our results demonstrate that distinct lineages have independently converged on a common molecular strategy to overcome the energetic barriers of ice formation. The discovery of cell-free INPs provides tools for freezing applications and reveals biophysical constraints on nucleation across life. 

Various aerosols, including mineral dust, soot, and biological particles, can act as ice nuclei, initiating the freezing of supercooled cloud droplets. Cloud droplet freezing significantly impacts cloud properties and, consequently, weather and climate. Some biological ice nuclei exhibit exceptionally high nucleation temperatures close to 0 °C. Ice-nucleating macromolecules (INMs) found on pollen are typically not considered among the most active ice nuclei. Still, they can be highly abundant, especially for species such as Betula pendula, a widespread birch tree species in the boreal forest. Recent studies have shown that certain tree-derived INMs exhibit ice nucleation activity above −10 °C, suggesting they could play a more significant role in atmospheric processes than previously understood. Our study reveals that three distinct INM classes active at −8.7, −15.7, and −17.4 °C are present in B. pendula. Freeze drying and freeze–thaw cycles noticeably alter their ice nucleation capability, and the results of heat treatment, size, and chemical analysis indicate that INM classes correspond to size-varying aggregates, with larger aggregates nucleating ice at higher temperatures, in agreement with previous studies on fungal and bacterial ice nucleators. Our findings suggest that B. pendula INMs are potentially important for atmospheric ice nucleation because of their high prevalence and nucleation temperatures. 

Abstract. From extracellular freezing to cloud glaciation, the crystallization of water is ubiquitous and shapes life as we know it. Efficient biological ice nucleators (INs) are crucial for organism survival in cold environments and, when aerosolized, serve as a significant source of atmospheric ice nuclei. Several lichen species have been identified as potent INs capable of inducing freezing at high subzero temperatures. Despite their importance, the abundance and diversity of lichen INs are still not well understood. Here, we investigate ice nucleation activity in the cyanolichen-forming genus Peltigera from across a range of ecosystems in the Arctic, the northwestern United States, and Central and South America. We find strong IN activity in all tested Peltigera species, with ice nucleation temperatures above −12 °C and 35 % of the samples initiating freezing at temperatures at or above −6.2 °C. The Peltigera INs in aqueous extract appear to be resistant to freeze–thaw cycles, suggesting that they can survive dispersal through the atmosphere and thereby potentially influence precipitation patterns. An axenic fungal culture termed L01-tf-B03, from the lichen Peltigera britannica JNU22, displays an ice nucleation temperature of −5.6 °C at 1 mg mL−1 and retains remarkably high IN activity at concentrations as low as 0.1 ng mL−1. Our analysis suggests that the INs released from this fungus in culture are 1000 times more potent than the most active bacterial INs from Pseudomonas syringae. The global distribution of Peltigera lichens, in combination with the IN activity, emphasizes their potential to act as powerful ice-nucleating agents in the atmosphere. 

Biological ice nucleation plays a key role in the survival of cold-adapted organisms. Several species of bacteria, fungi, and insects produce ice nucleators (INs) that enable ice formation at temperatures above −10 °C. Bacteria and fungi produce particularly potent INs that can promote water crystallization above −5 °C. Bacterial INs consist of extended protein units that aggregate to achieve superior functionality. Despite decades of research, the nature and identity of fungal INs remain elusive. Here, we combine ice nucleation measurements, physicochemical characterization, numerical modeling, and nucleation theory to shed light on the size and nature of the INs from the fungusFusarium acuminatum. We find ice-binding and ice-shaping activity ofFusariumIN, suggesting a potential connection between ice growth promotion and inhibition. We demonstrate that fungal INs are composed of small 5.3 kDa protein subunits that assemble into ice-nucleating complexes that can contain more than 100 subunits.FusariumINs retain high ice-nucleation activity even when only the ~12 kDa fraction of size-excluded proteins are initially present, suggesting robust pathways for their functional aggregation in cell-free aqueous environments. We conclude that the use of small proteins to build large assemblies is a common strategy among organisms to create potent biological INs. 

Abstract. Forty years ago, lichens were identified as extraordinary biological icenucleators (INs) that enable ice formation at temperatures close to0 ∘C. By employing INs, lichens thrive in freezing environmentsthat surpass the physiological limits of other vegetation, thus making themthe majority of vegetative biomass in northern ecosystems. Aerosolizedlichen INs might further impact cloud glaciation and have the potential toalter atmospheric processes in a warming Arctic. Despite the ecologicalimportance and formidable ice nucleation activities, the abundance,diversity, sources, and role of ice nucleation in lichens remain poorlyunderstood. Here, we investigate the ice nucleation capabilities of lichenscollected from various ecosystems across Alaska. We find ice nucleatingactivity in lichen to be widespread, particularly in the coastal rainforestof southeast Alaska. Across 29 investigated lichen, all species show icenucleation temperatures above −15 ∘C, and ∼30 %initiate freezing at temperatures above −6 ∘C. Concentrationseries of lichen ice nucleation assays in combination with statisticalanalysis reveal that the lichens contain two subpopulations of INs, similarto previous observations in bacteria. However, unlike the bacterial INs, thelichen INs appear as independent subpopulations resistant to freeze–thawcycles and against temperature treatment. The ubiquity and high stability ofthe lichen INs suggest that they can impact local atmospheric processes andthat ice nucleation activity is an essential trait for their survival incold environments. 

The heterogeneous nucleation of ice is an importantatmospheric process facilitated by a wide range of aerosols. Drop-freezingexperiments are key for the determination of the ice nucleation activity ofbiotic and abiotic ice nucleators (INs). The results of these experimentsare reported as the fraction of frozen droplets fice(T) as a functionof decreasing temperature and the corresponding cumulative freezing spectraNm(T) computed using Gabor Vali's methodology. The differential freezingspectrum nm(T) is an approximant to the underlying distribution ofheterogeneous ice nucleation temperatures Pu(T) that represents thecharacteristic freezing temperatures of all INs in the sample. However,Nm(T) can be noisy, resulting in a differential form nm(T) that is challenging to interpret. Furthermore, there is no rigorousstatistical analysis of how many droplets and dilutions are needed to obtaina well-converged nm(T) that represents the underlying distributionPu(T). Here, we present the HUB (heterogeneousunderlying-based) method and associated Python codes thatmodel (HUB-forward code) and interpret (HUB-backward code) the results ofdrop-freezing experiments. HUB-forward predicts fice(T) and Nm(T)from a proposed distribution Pu(T) of IN temperatures, allowing itsusers to test hypotheses regarding the role of subpopulations of nuclei infreezing spectra and providing a guide for a more efficient collection offreezing data. HUB-backward uses a stochastic optimization method to computenm(T) from either Nm(T) or fice(T). The differential spectrumcomputed with HUB-backward is an analytical function that can be used toreveal and characterize the underlying number of IN subpopulations ofcomplex biological samples (e.g., ice-nucleating bacteria, fungi, pollen)and to quantify the dependence of these subpopulations on environmentalvariables. By delivering a way to compute the differential spectrum fromdrop-freezing data, and vice versa, the HUB-forward and HUB-backward codesprovide a hub to connect experiments and interpretative physical quantitiesthat can be analyzed with kinetic models and nucleation theory.