High Ice Nucleation Activity Research Articles

High interspecific variability in ice nucleation activity suggests pollen ice nucleators are incidental

Abstract. Ice-nucleating macromolecules (INMs) produced by plant pollen can nucleate ice at warm temperatures and may play an important role in weather- and climate-relevant cloud glaciation. INMs have also proved useful for mammalian cell and tissue model cryopreservation. The high ice nucleation (IN) activity of some INMs indicates an underlying biological function, either freezing tolerance or bioprecipitation-mediated dispersal. Here, using the largest study of pollen ice nucleation to date, we show that phylogenetic proximity, spermatophyte subdivision, primary growth biome, pollination season, primary pollination method, desiccation tolerance and native growth elevation do not account for the IN activity of INMs released from different plant species' pollen. The results suggest that these macromolecules are produced by plants for a purpose unrelated to ice nucleation and have an incidental ability to nucleate ice. This ability may have been adapted by some species for specific biological purposes, producing exceptional ice nucleators. Pollen INMs may be more active, widespread in nature, and diverse than previously thought.

High-speed cryo-microscopy reveals that ice-nucleating proteins of Pseudomonas syringae trigger freezing at hydrophobic interfaces.

Ice-nucleating proteins (INpro) trigger the freezing of supercooled water droplets relevant to atmospheric, biological, and technological applications. The high ice nucleation activity of INpro isolated from the bacteria Pseudomonas syringae could be linked to the aggregation of proteins at the bacterial membrane or at the air-water interface (AWI) of droplets. Here, we imaged freezing onsets, providing direct evidence of these proposed mechanisms. High-speed cryo-microscopy identified the onset location of freezing in droplets between two protein-repellent glass slides. INpro from sterilized P. syringae (Snomax) statistically favored nucleation at the AWI of the droplets. Removing cellular fragments by filtration or adding surfactants increased the frequency of nucleation events at the AWI. On the other hand, cultivated intact bacteria cells or lipid-free droplets nucleated ice without an affinity to the AWI. Overall, we provide visual evidence that INpro from P. syringae trigger freezing at hydrophobic interfaces, such as the AWI or the bacterial membrane, with important mechanistic implications for applications of INpro.

Functional aggregation of cell-free proteins enables fungal ice nucleation.

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 fungus Fusarium acuminatum. We find ice-binding and ice-shaping activity of Fusarium IN, 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. Fusarium INs 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.

K-feldspar enrichment in the Pacific pelagic sediments before Miocene

The mineralogy of atmospheric silicate dust controls its interaction with clouds. K-feldspar has a remarkably high ice-nucleating activity, and its distribution may have influenced the global climate throughout Earth's history. However, long-term archives of past atmospheric feldspar are not known. Here, we investigate feldspar mineralogy, content, and grain size in pelagic clay cores. Sediments around Minamitorishima Island contain > 10 wt% of K-feldspar before ~ 35 Ma, which is five times more than the younger sediments. This distribution does not resemble other volcanic minerals or geochemically estimated volcanic input, suggesting that the K-feldspars are not associated with volcanic ash. The K-feldspars are present as isolated grains as well as pseudorhombohedral microcrystals indicative of authigenic overgrowth. On the other hand, they contain some Na, arguing against a purely authigenic origin. Grain size distributions of chemically separated quartz and feldspars show stratigraphic variation analogous to other North Pacific sites, further suggesting a link to eolian materials. Sediments from a South Pacific site also show K-feldspar enrichment over plagioclase before ~ 44 Ma, although the content relative to bulk sediment does not change much. We propose that the K-feldspar may be enriched in the wide area of the Pacific before ~ 30 to 40 Ma.

Toward Understanding Bacterial Ice Nucleation.

Bacterial ice nucleators (INs) are among the most effective ice nucleators known and are relevant for freezing processes in agriculture, the atmosphere, and the biosphere. Their ability to facilitate ice formation is due to specialized ice-nucleating proteins (INPs) anchored to the outer bacterial cell membrane, enabling the crystallization of water at temperatures up to −2 °C. In this Perspective, we highlight the importance of functional aggregation of INPs for the exceptionally high ice nucleation activity of bacterial ice nucleators. We emphasize that the bacterial cell membrane, as well as environmental conditions, is crucial for a precise functional INP aggregation. Interdisciplinary approaches combining high-throughput droplet freezing assays with advanced physicochemical tools and protein biochemistry are needed to link changes in protein structure or protein–water interactions with changes on the functional level.

Different characteristics of microbial diversity and special functional microbes in rainwater and topsoil before and after 2019 new coronavirus epidemic in Inner Mongolia Grassland

Grassland ecosystems are vital terrestrial ecosystems. As areas sensitive to climate change, they are critical for assessing the effects of global climate change. In China, grasslands account for over 40% of the land area. There is currently limited information on microbial diversity evolution in different grassland areas, particularly microorganisms with ice nucleation activity (INA) and their potential resources with potential influence to regulate regional precipitation and climate. We used Illumina MiSeq to sequence the 16S rRNA V3–V4 hypervariable region and performed a simple droplet freezing experiment to determine the variation in the grassland microbial community species composition and community structure. Rainwater and topsoil samples from the Hulunbuir Grassland in Inner Mongolia collected over three years were characterized. The dominant bacterial genus in the rainwater was Massilia, and the dominant fungus was Cladosporium. Additionally, the dominant bacteria in the soil were Sphingomonas, and the dominant fungus was Gibberella. There were differences in the microbial communities before and after the coronavirus disease epidemic. Pathogenic microorganisms exhibited inconsistent responses to environmental changes. The low relative abundance of known high-INA microorganisms and the higher freezing temperature indicated that unknown high-efficiency biological ice nucleating particles may be present. We found significant differences in species diversity and richness between the rainwater and soil populations in grassland areas by analyzing the sample community structures. Our research results revealed the species composition and structure of the microbiota in grassland ecosystems in China, indicating that environmental media and human activities may affect the microbiota in the grassland area and indicating underlying microorganisms with high INA.

Effect of chemically induced fracturing on the ice nucleation activity of alkali feldspar

Abstract. Feldspar is an important constituent of airborne mineral dust. Some alkali feldspars exhibit particularly high ice nucleation (IN) activity. This has been related to structural similarities of the ice (101‾0) prism planes and the (100) planes of alkali feldspar. Here the effect of generating feldspar surfaces with close to (100) orientation by means of chemically induced fracturing on the IN activity of alkali feldspar was investigated experimentally. To this end, gem-quality K-rich alkali feldspar was shifted towards more Na-rich compositions by cation exchange with an NaCl–KCl salt melt at 850 ∘C. By this procedure, a system of parallel cracks with an orientation close to the (100) plane of the feldspar was induced. Droplet-freezing assay experiments performed on grain mounts of the cation-exchanged alkali feldspars revealed an increase in the overall density of ice-nucleating active site (INAS) density with respect to the untreated feldspar. In addition, annealing at 550 ∘C subsequent to primary cation exchange further enhanced the INAS density and led to IN activity at exceptionally high temperatures. Although very efficient in experiment, fracturing by cation exchange with an alkali halide salt is unlikely to be of relevance in the conditioning of alkali feldspars in nature. However, parting planes with similar orientation as the chemically induced cracks may be generated in lamellar microstructures resulting from the exsolution of initially homogeneous alkali feldspar, a widespread phenomenon in natural alkali feldspar known as perthite formation. Perthitic alkali feldspars indeed show the highest IN activity. We tentatively ascribe this phenomenon to the preferential exposure of feldspar crystal surfaces oriented sub-parallel to (100).

Factors contributing to ice nucleation and sequential freezing of leaves in wheat

Main conclusionAnatomical, metabolic and microbial factors were identified that contribute to sequential freezing in wheat leaves and likely contribute to supercooling in the youngest leaves and potentially meristematic regions.Infrared thermography (IR) has been used to observe wheat leaves freezing independently and in an age-related sequence with older leaves freezing first. To determine mechanisms that might explain this sequence of freezing several analytical approaches were used: (1) The size of xylem vessels, in proximity to where freezing initiated, was measured to see if capillary freezing point depression explained sequential freezing. The sequence of freezing in the four youngest leaves was correlated, with the largest vessels freezing first. (2) Carbohydrate and amino acids were analyzed to determine if solute concentrations as well as interactions with membranes explained the freezing sequence. Sucrose was highly correlated to the freezing sequence for all leaves suggesting a prominent role for this sugar as compared to other simple sugars and fructans. Among individual free amino acids proline and serine were correlated to the freezing sequence, with younger leaves having the highest concentrations. (3) Microflora within and on leaf surfaces were determined to measure potential freezing initiation. Levels of bacteria and fungi were correlated to the freezing sequence for all leaves, and species or genera associated with high ice nucleation activity were absent in younger leaves. Moisture content and transcript expression of ice binding proteins were also measured. As expected, our results show that no single mechanism explains the freezing sequence observed via infrared analyses. While these multiple mechanisms are operative at different levels according to the leaf age, they seem to converge when it comes to the protection of vital meristematic tissues. This provides potential phenotypic characters that could be used by breeders to develop more winter-hardy genotypes.

Ice nucleation activity of iron oxides via immersion freezing and an examination of the high ice nucleation activity of FeO.

Heterogeneous ice nucleation is a common process in the atmosphere, but relatively little is known about the role of different surface characteristics on the promotion of ice nucleation. We have used a series of iron oxides as a model system to study the role of lattice mismatch and defects induced by milling on ice nucleation activity. The iron oxides include wüstite (FeO), hematite (Fe2O3), magnetite (Fe3O4), and goethite (FeOOH). The iron oxides were characterized by X-ray diffraction (XRD) and Brunauer-Emmett-Teller (BET) surface area measurements. The immersion freezing experiments were performed using an environmental chamber. Wüstite (FeO) had the highest ice nucleation activity, which we attribute to its low lattice mismatch with hexagonal ice and the exposure of Fe-OH after milling. A comparison study of MnO and wüstite (FeO) with milled and sieved samples for each suggests that physical defects alone result in only a slight increase in ice nucleation activity. Despite differences in the molecular formula and surface groups, hematite (Fe2O3), magnetite (Fe3O4), and goethite (FeOOH) had similar ice nucleation activities, which may be attributed to their high lattice mismatch to hexagonal ice. This study provides further insight into the characteristics of a good heterogeneous ice nucleus and, more generally, helps to elucidate the interactions between aerosol particles and ice particles in clouds.

Interfacial Water Ordering Is Insufficient to Explain Ice-Nucleating Protein Activity.

Ice-nucleating proteins (INPs) found in bacteria are the most effective ice nucleators known, enabling the crystallization of water at temperatures close to 0 °C. Although their function has been known for decades, the underlying mechanism is still under debate. Here, we show that INPs from Pseudomonas syringae in aqueous solution exhibit a defined solution structure and show no significant conformational changes upon cooling. In contrast, irreversible structural changes are observed upon heating to temperatures exceeding ∼55 °C, leading to a loss of the ice-nucleation activity. Sum-frequency generation (SFG) spectroscopy reveals that active and heat-inactivated INPs impose similar structural ordering of interfacial water molecules upon cooling. Our results demonstrate that increased water ordering is not sufficient to explain INPs' high ice-nucleation activity and confirm that intact three-dimensional protein structures are critical for bacterial ice nucleation, supporting a mechanism that depends on the INPs' supramolecular interactions.

Glucose as a Potential Chemical Marker for Ice Nucleating Activity in Arctic Seawater and Melt Pond Samples.

Recent studies pointed to a high ice nucleating activity (INA) in the Arctic sea surface microlayer (SML). However, related chemical information is still sparse. In the present study, INA and free glucose concentrations were quantified in Arctic SML and bulk water samples from the marginal ice zone, the ice-free ocean, melt ponds, and open waters within the ice pack. T50 (defining INA) ranged from -17.4 to -26.8 °C. Glucose concentrations varied from 0.6 to 51 μg/L with highest values in the SML from the marginal ice zone and melt ponds (median 16.3 and 13.5 μg/L) and lower values in the SML from the ice pack and the ice-free ocean (median 3.9 and 4.0 μg/L). Enrichment factors between the SML and the bulk ranged from 0.4 to 17. A positive correlation was observed between free glucose concentration and INA in Arctic water samples (T50(°C) = (-25.6 ± 0.6) + (0.15 ± 0.04)·Glucose(μg/L), RP = 0.66, n = 74). Clustering water samples based on phytoplankton pigment composition resulted in robust but different correlations within the four clusters (RP between 0.67 and 0.96), indicating a strong link to phytoplankton-related processes. Since glucose did not show significant INA itself, free glucose may serve as a potential tracer for INA in Arctic water samples.

The importance of crystalline phases in ice nucleation by volcanic ash

Abstract. Volcanic ash is known to nucleate ice when immersed in supercooled water droplets. This process may impact the properties and dynamics of the eruption plume and cloud as well as those of meteorological clouds once the ash is dispersed in the atmosphere. However, knowledge of what controls the ice-nucleating activity (INA) of ash remains limited, although it has been suggested that crystalline components in ash may play an important role. Here we adopted a novel approach using nine pairs of tephra and their remelted and quenched glass equivalents to investigate the influence of chemical composition, crystallinity, and mineralogy on ash INA in the immersion mode. For all nine pairs studied, the crystal-bearing tephra nucleated ice at warmer temperatures than the corresponding crystal-free glass, indicating that crystalline phases are key to ash INA. Similar to findings for desert dust from arid and semi-arid regions, the presence of feldspar minerals characterizes the four most ice-active tephra samples, although a high INA is observed even in the absence of alkali feldspar in samples bearing plagioclase feldspar and orthopyroxene. There is evidence of a potential indirect relationship between chemical composition and ash INA, whereby a magma of felsic to intermediate composition may generate ash containing ice-active feldspar or pyroxene minerals. This complex interplay between chemical composition, crystallinity, and mineralogy could help to explain the variability in volcanic ash INA reported in the literature. Overall, by demonstrating the importance of crystalline phases in the INA of ash, our study contributes insights essential for better appraising the role of airborne ash in ice formation. Among these is the inference that glass-dominated ash emitted by the largest explosive volcanic eruptions might be less effective at impacting ice-nucleating particle populations than crystalline ash generated by smaller, more frequent eruptions.

The Effect of Crystallinity and Crystal Structure on the Immersion Freezing of Alumina.

Determining the factors that constitute an efficient ice nucleus is an ongoing area of research in the atmospheric community. In particular, surface characteristics such as functional groups and surface defects impact the ice nucleation efficiency. Crystal structure has been proposed to be a possible factor that can dictate ice nucleation activity through the templating of water molecules on the surface of the aerosol particle. If the crystal structure of the surface matches that of the crystal structure of ice, it has been shown to increase ice nucleation activity. In this study, alumina was chosen as a model system because crystal structure and crystallinity can be tuned, and the effect on immersion freezing was explored. The nine alumina samples include polymorphs of AlOOH, Al(OH)3, and Al2O3, which have a range of crystal structures and crystallinities. The samples were characterized with X-ray diffraction (XRD), scanning electron microscopy (SEM), transmission electron microscopy (TEM), energy dispersive spectroscopy (EDS), and Brunauer-Emmett-Teller (BET) analysis. From the immersion freezing experiments, corundum [α-Al2O3] was shown to have the highest ice nucleation activity likely because of its high lattice match and high degree of crystallinity. Crystal structure alone did not show a strong correlation with ice nucleation activity, but a combination of a hexagonal crystal structure and a highly crystalline surface was seen to nucleate ice at warmer temperatures than the other alumina samples. This study provides experimental results in the study of ice nucleation of a range of alumina samples, which have possible implications for alumina-based mineral dust particles. Our findings suggest that crystallinity and crystal structure are important to consider when evaluating the ice nucleation efficiency of aerosol particles in laboratory and modeling studies.

Ice segregation in the crown of winter cereals: Evidence for extraorgan and extratissue freezing

Meaningful improvements in winter cereal cold hardiness requires a complete model of freezing behaviour in the critical crown organ. Magnetic resonance microimaging diffusion-weighted experiments provided evidence that cold acclimation decreased water content and mobility in the vascular transition zone (VTZ) and the intermediate zone in rye (Secale cereale L. Hazlet) compared with wheat (Triticum aestivum L. Norstar). Differential thermal analysis, ice nucleation, and localization studies identified three distinct exothermic events. A high-temperature exotherm (-3°C to -5°C) corresponded with ice formation and high ice-nucleating activity in the leaf sheath encapsulating the crown. A midtemperature exotherm (-6°C and -8°C) corresponded with cavity ice formation in the VTZ but an absence of ice in the shoot apical meristem (SAM). A low-temperature exotherm corresponded with SAM injury and the killing temperature in wheat (-21°C) and rye (-27°C). The SAM had lower ice-nucleating activity and freezing survival compared with the VTZ when frozen in vitro. The intermediate zone was hypothesized to act as a barrier to ice growth into the SAM. Higher cold hardiness of rye compared with wheat was associated with higher VTZ and intermediate zone desiccation resulting in the formation of ice barriers surrounding the SAM.

Ice Nucleation Activity in Plants: The Distribution, Characterization, and Their Roles in Cold Hardiness Mechanisms.

Control of freezing in plant tissues is a key issue in cold hardiness mechanisms. Yet freeze-regulation mechanisms remain mostly unexplored. Among them, ice nucleation activity (INA) is a primary factor involved in the initiation and regulation of freezing events in plant tissues, yet the details remain poorly understood. To address this, we developed a highly reproducible assay for determining plant tissue INA and noninvasive freeze visualization tools using MRI and infrared thermography. The results of visualization studies on plant freezing behaviors and INA survey of over 600 species tissues show that (1) freezing-sensitive plants tend to have low INA in their tissues (thus tend to transiently supercool), while wintering cold-hardy species have high INA in some specialized tissues; and (2) the high INA in cold-hardy tissues likely functions as a freezing sensor to initiate freezing at warm subzero temperatures at appropriate locations and timing, resulting in the induction of tissue-/species-specific freezing behaviors (e.g., extracellular freezing, extraorgan freezing) and the freezing order among tissues: from the primary freeze to the last tissue remaining unfrozen (likely INA level dependent). The spatiotemporal distributions of tissue INA, their characterization, and functional roles are detailed. INA assay principles, anti-nucleation activity (ANA), and freeze visualization tools are also described.

Heterogeneous ice nucleation on dust particles sourced from nine deserts worldwide – Part 1: Immersion freezing

Abstract. Desert dust is one of the most abundant ice nucleating particle types in the atmosphere. Traditionally, clay minerals were assumed to determine the ice nucleation ability of desert dust and constituted the focus of ice nucleation studies over several decades. Recently some feldspar species were identified to be ice active at much higher temperatures than clay minerals, redirecting studies to investigate the contribution of feldspar to ice nucleation on desert dust. However, so far no study has shown the atmospheric relevance of this mineral phase.For this study four dust samples were collected after airborne transport in the troposphere from the Sahara to different locations (Crete, the Peloponnese, Canary Islands, and the Sinai Peninsula). Additionally, 11 dust samples were collected from the surface from nine of the biggest deserts worldwide. The samples were used to study the ice nucleation behavior specific to different desert dusts. Furthermore, we investigated how representative surface-collected dust is for the atmosphere by comparing to the ice nucleation activity of the airborne samples. We used the IMCA-ZINC setup to form droplets on single aerosol particles which were subsequently exposed to temperatures between 233 and 250 K. Dust particles were collected in parallel on filters for offline cold-stage ice nucleation experiments at 253–263 K. To help the interpretation of the ice nucleation experiments the mineralogical composition of the dusts was investigated. We find that a higher ice nucleation activity in a given sample at 253 K can be attributed to the K-feldspar content present in this sample, whereas at temperatures between 238 and 245 K it is attributed to the sum of feldspar and quartz content present. A high clay content, in contrast, is associated with lower ice nucleation activity. This confirms the importance of feldspar above 250 K and the role of quartz and feldspars determining the ice nucleation activities at lower temperatures as found by earlier studies for monomineral dusts. The airborne samples show on average a lower ice nucleation activity than the surface-collected ones. Furthermore, we find that under certain conditions milling can lead to a decrease in the ice nucleation ability of polymineral samples due to the different hardness and cleavage of individual mineral phases causing an increase of minerals with low ice nucleation ability in the atmospherically relevant size fraction. Comparison of our data set to an existing desert dust parameterization confirms its applicability for climate models. Our results suggest that for an improved prediction of the ice nucleation ability of desert dust in the atmosphere, the modeling of emission and atmospheric transport of the feldspar and quartz mineral phases would be key, while other minerals are only of minor importance.

Ice nucleation activity in various tissues of Rhododendron flower buds: their relevance to extraorgan freezing.

Wintering flower buds of cold hardy Rhododendron japonicum cooled slowly to subfreezing temperatures are known to undergo extraorgan freezing, whose mechanisms remain obscure. We revisited this material to demonstrate why bud scales freeze first in spite of their lower water content, why florets remain deeply supercooled and how seasonal adaptive responses occur in regard to extraorgan freezing in flower buds. We determined ice nucleation activity (INA) of various flower bud tissues using a test tube-based assay. Irrespective of collection sites, outer and inner bud scales that function as ice sinks in extraorgan freezing had high INA levels whilst florets that remain supercooled and act as a water source lacked INA. The INA level of bud scales was not high in late August when flower bud formation was ending, but increased to reach the highest level in late October just before the first autumnal freeze. The results support the following hypothesis: the high INA in bud scales functions as the subfreezing sensor, ensuring the primary freezing in bud scales at warmer subzero temperatures, which likely allows the migration of floret water to the bud scales and accumulation of icicles within the bud scales. The low INA in the florets helps them remain unfrozen by deep supercooling. The INA in the bud scales was resistant to grinding and autoclaving at 121∘C for 15 min, implying the intrinsic nature of the INA rather than of microbial origin, whilst the INA in stem bark was autoclaving-labile. Anti-nucleation activity (ANA) was implicated in the leachate of autoclaved bud scales, which suppresses the INA at millimolar levels of concentration and likely differs from the colligative effects of the solutes. The tissue INA levels likely contribute to the establishment of freezing behaviors by ensuring the order of freezing in the tissues: from the primary freeze to the last tissue remaining unfrozen.

High ice nucleation activity located in blueberry stem bark is linked to primary freeze initiation and adaptive freezing behaviour of the bark.

Controlled ice nucleation is an important mechanism in cold-hardy plant tissues for avoiding excessive supercooling of the protoplasm, for inducing extracellular freezing and/or for accommodating ice crystals in specific tissues. To understand its nature, it is necessary to characterize the ice nucleation activity (INA), defined as the ability of a tissue to induce heterogeneous ice nucleation. Few studies have addressed the precise localization of INA in wintering plant tissues in respect of its function. For this purpose, we recently revised a test tube INA assay and examined INA in various tissues of over 600 species. Extremely high levels of INA (-1 to -4 °C) in two wintering blueberry cultivars of contrasting freezing tolerance were found. Their INA was much greater than in other cold-hardy species and was found to be evenly distributed along the stems of the current year's growth. Concentrations of active ice nuclei in the stem were estimated from quantitative analyses. Stem INA was localized mainly in the bark while the xylem and pith had much lower INA. Bark INA was located mostly in the cell wall fraction (cell walls and intercellular structural components). Intracellular fractions had much less INA. Some cultivar differences were identified. The results corresponded closely with the intrinsic freezing behaviour (extracellular freezing) of the bark, icicle accumulation in the bark and initial ice nucleation in the stem under dry surface conditions. Stem INA was resistant to various antimicrobial treatments. These properties and specific localization imply that high INA in blueberry stems is of intrinsic origin and contributes to the spontaneous initiation of freezing in extracellular spaces of the bark by acting as a subfreezing temperature sensor.

Expression and localization of an ice nucleating protein from a soil bacterium, Pseudomonas borealis

An ice nucleating protein (INP) coding region with 66% sequence identity to the INP of Pseudomonas syringae was previously cloned from P. borealis, a plant beneficial soil bacterium. Ice nucleating activity (INA) in the P. borealis DL7 strain was highest after transfer of cultures to temperatures just above freezing. The corresponding INP coding sequence (inaPb or ina) was used to construct recombinant plasmids, with recombinant expression visualized using a green fluorescent protein marker (gfp encoding GFP). Although the P. borealis strain was originally isolated by ice-affinity, bacterial cultures with membrane-associated INP-GFP did not adsorb to pre-formed ice. Employment of a shuttle vector allowed expression of ina-gfp in both Escherichia coli and Pseudomonas cells. At 27°C, diffuse fluorescence appeared throughout the cells and was associated with low INA. However, after transfer of cultures to 4°C, the protein localized to the poles coincident with high INA. Transformants with truncated INP sequences ligated to either gfp, or an antifreeze protein-gfp fusion showed that the repetitive ice-nucleation domain was not necessary for localization. Such localization is consistent with the flanking residues of the INP associating with a temperature-dependent secretion apparatus. A polar location would facilitate INP–INP interactions resulting in the formation of larger aggregates, serving to increase INA. Expression of INPs by P. borealis could function as an efficient atmospheric dispersal mechanism for these soil bacteria, which are less likely to use these proteins for nutrient procurement, as has been suggested for P. syringae.

Seasonal changes in ice nucleation activity in blueberry stems and effects of cold treatments in vitro

Ice nucleation activity (INA) of plant intrinsic origins is considered to play important roles in plant cold hardiness mechanisms. Yet, only a few studies have addressed the spatial and temporal localization of plant INA, how it is regulated and what its functional roles are. In our previous study (Kishimoto et al., 2014), we revised a test tube method and developed a highly reproducible assay for measuring INA of plant specimens and demonstrated that high INA occurred in the cell wall fraction of wintering bark tissues of blueberry stems and corresponded well to the freezing behavior (extracellular freezing) of the stem bark. Here, we followed precisely seasonal changes in the stem INA of two blueberry cultivars and alterations in the stem INA caused by artificial incubation at various low temperatures. INA of newly developed shoots was low but increased rapidly by July when the stem became seemingly matured, then gradually increased with the maximum in October or early November just before the first autumnal frost. Following the subsequent recurrent frosts, the stem INA gradually decreased. This tendency was consistent between the two cultivars differing in the level of cold hardiness. INA in the stems of September until February was increased by incubation at 0-7°C whilst decreased by freezing to lower temperatures. The in vitro results corroborate the seasonal changes in the stem INA in the field but the mechanisms remain to be investigated. The highest level of INA (expressed as the median ice nucleation temperature) observed with current year stems (7.5 mm-long) of Woodard in October of 2010-2013 was -0.9 ∼ -1.0°C when determined with 2mL assay system (-1.1 ∼ -1.3°C with 0.5mL system). This may likely be one of the highest INA of biological origins ever reported.