scholarly journals Characterization of Chromatin Accessibility and Gene Expression Upon Cold Stress Reveals the Transcription Factor RAV1 Functions in Cold Response in Vitis amurensis

2021 ◽  
Author(s):  
Chong Ren ◽  
Li Huayang ◽  
Zemin Wang ◽  
Zhanwu Dai ◽  
Fatma Lecourieux ◽  
...  
Keyword(s):  
Cold Stress ◽  
Cold Tolerance ◽  
Regulatory Networks ◽  
Cold Hardiness ◽  
Target Genes ◽  
Plant Cell Wall ◽  
Cold Treatment ◽  
Vitis Amurensis ◽  
Rna Seq ◽  
Cold Response

Abstract Cold tolerance is regulated by a variety of transcription factors (TFs) and their target genes. Except for the well-characterized C-repeat binding factors (CBFs)-dependent transcriptional cascade, the mechanisms of cold tolerance mediated by other transcriptional regulatory networks are still largely unknown. Here we used the assay for transposase-accessible chromatin with sequencing (ATAC-seq) and RNA-seq to identify cold responsive TFs in Vitis amurensis, a grape species with high cold hardiness. A number of 9 TFs, including CBF4, RAV1 and ERF104, were identified after cold treatment. Weighted gene co-expression network analysis (WGCNA) and gene ontology (GO) analysis revealed that these TFs may regulate cold response through different pathways. As a prime candidate TF, overexpression of VaRAV1 in grape cells improved its cold tolerance. The transgenic cells exhibited low electrolyte leakage and malondialdehyde (MDA) content and high peroxidase (POD) activity. Moreover, the TF gene TCP8 and a gene involving in homogalacturonan biosynthesis were found to be regulated by VaRAV1, suggesting that the contribution of VaRAV1 to cold tolerance may be achieved by enhancing stability of cell membrane and regulating the expression of target genes involved in plant cell wall composition. Our work provides novel insights into plant response to cold stress and demonstrates the utility of ATAC-seq and RNA-seq for rapid identification of TFs in response to cold stress in grapevine. The VaRAV1 may play an important role in adaption to cold stress.

GRAS-domain transcription factor PAT1 regulates jasmonic acid biosynthesis in grape cold stress response

2021 ◽  
Author(s):  
Zemin Wang ◽  
Darren Chern Jan Wong ◽  
Yi Wang ◽  
Guangzhao Xu ◽  
Chong Ren ◽  
...  
Keyword(s):  
Stress Response ◽  
Cold Stress ◽  
Cold Tolerance ◽  
Cold Treatment ◽  
Ectopic Expression ◽  
Bimolecular Fluorescence Complementation ◽  
Luciferase Reporter ◽  
Vitis Amurensis ◽  
Mobility Shift

Abstract Cultivated grapevine (Vitis) is a highly valued horticultural crop, and cold stress affects its growth and productivity. Wild Amur grape (Vitis amurensis) PAT1 (Phytochrome A signal transduction 1, VaPAT1) is induced by low temperature, and ectopic expression of VaPAT1 enhances cold tolerance in Arabidopsis (Arabidopsis thaliana). However, little is known about the molecular mechanism of VaPAT1 during the cold stress response in grapevine. Here, we confirmed the overexpression of VaPAT1 in transformed grape calli enhanced cold tolerance. Yeast two-hybrid and bimolecular fluorescence complementation assays highlighted an interaction between VaPAT1 with INDETERMINATE-DOMAIN 3 (VaIDD3). A role of VaIDD3 in cold tolerance was also indicated. Transcriptome analysis revealed VaPAT1 and VaIDD3 overexpression and cold treatment coordinately modulate the expression of stress-related genes including lipoxygenase 3 (LOX3), a gene encoding a key jasmonate biosynthesis enzyme. Co-expression network analysis indicated LOX3 might be a downstream target of VaPAT1. Both electrophoretic mobility shift and dual luciferase reporter assays showed the VaPAT1-IDD3 complex binds to the IDD-box (AGACAAA) in the VaLOX3 promoter to activate its expression. Overexpression of both VaPAT1 and VaIDD3 increased the transcription of VaLOX3 and JA levels in transgenic grape calli. Conversely, VaPAT1-SRDX (dominant repression) and CRISPR/Cas9-mediated mutagenesis of PAT1-ED causing the loss of the C-terminus in grape calli dramatically prohibited the accumulation of VaLOX3 and JA levels during cold treatment. Together, these findings point to a pivotal role of VaPAT1 in the cold stress response in grape by regulating JA biosynthesis.

scholarly journals iTRAQ-based quantitative proteomics analysis of cantaloupe (Cucumis melo var. saccharinus) after cold storage

2020 ◽  
Author(s):  
Wen Song ◽  
Fengxian Tang ◽  
Wenchao Cai ◽  
Qin Zhang ◽  
Fake Zhou ◽  
...  
Keyword(s):  
Cold Stress ◽  
Cold Tolerance ◽  
Proteomic Analysis ◽  
Molecular Mechanisms ◽  
Treatment Time ◽  
Chilling Injury ◽  
Cold Treatment ◽  
Cold Response ◽  
Low Temperature Storage ◽  
Metabolic Analysis

Abstract Background: During the low temperature storage, cantaloupe is susceptible to the cold stress, resulting in the loss of edible and commercial quality. To ascertain the molecular mechanisms of cold tolerance in cantaloupe, cold-sensitive cultivar Goldqueen-308 (GE) and cold-tolerant cultivar Jiashi-310 (JS) were used for quantitative proteomic analysis with iTRAQ in parallel. Results: In this work, two commercial cultivars were treated at 0.5℃ for 0, 12 and 24 days. Phenotypes assays showed that GE suffered a more severe damage as the cold treatment time extended. Proteomic analysis revealed that the number of differentially expressed proteins (DEPs) changed significantly over time in cold-exposed cantaloupe. Comparing with GE, JS responded in a prompter manner in terms of expressing cold-responding proteins during the similarly cold treatment. Furthermore, much more different groups of proteins were mobilized in response to the cold treatment in JS comparing with GE. Metabolic analysis indicated that more amino acids were up-regulated in JS during the early phases of cold stress. This study also identified some DEPs since they were up-regulated in JS or down-regulated in GE in terms of molecular mechanisms, which were mainly related to carbohydrate and energy metabolism, structure proteins, ROS scavening, amino acid metabolic and signaling transduction. Moreover, iTRAQ analysis was confirmed to be reliable via the results of phenotypes assays, metabolic analysis and q-PCR validation. Conclusion: By proteomics information,we found that the prompt response and the significant mobilization of proteins in JS maintained a higher level of cold tolerance, and the delay of cold response in GE could be a critical reason for the severe chilling injury. The candidate proteins we found will be the basis of future studies for further investigations and our findings may help to better understand the novel mechanisms of cold tolerance in cantaloupe.

scholarly journals iTRAQ-based quantitative proteomics analysis of cantaloupe (Cucumis melo var. saccharinus) after cold storage

2020 ◽  
Author(s):  
Wen Song ◽  
Fengxian Tang ◽  
Wenchao Cai ◽  
Qin Zhang ◽  
Fake Zhou ◽  
...  
Keyword(s):  
Cold Stress ◽  
Cold Tolerance ◽  
Proteomic Analysis ◽  
Molecular Mechanisms ◽  
Treatment Time ◽  
Chilling Injury ◽  
Cold Treatment ◽  
Cold Response ◽  
Low Temperature Storage ◽  
Metabolic Analysis

Abstract Background: During the low temperature storage, cantaloupe is susceptible to the cold stress, resulting in the loss of edible and commercial quality. To ascertain the molecular mechanisms of cold tolerance in cantaloupe, cold-sensitive cultivar Goldqueen-308 (GE) and cold-tolerant cultivar Jiashi-310 (JS) were used for quantitative proteomic analysis with iTRAQ in parallel. Results: In this work, two commercial cultivars were treated at 0.5℃ for 0, 12 and 24 days. Phenotypes assays showed that GE suffered a more severe damage as the cold treatment time extended. Proteomic analysis revealed that the number of differentially expressed proteins (DEPs) changed significantly over time in cold-exposed cantaloupe. Comparing with GE, JS responded in a prompter manner in terms of expressing cold-responding proteins during the similarly cold treatment. Furthermore, much more different groups of proteins were mobilized in response to the cold treatment in JS comparing with GE. Metabolic analysis indicated that more amino acids were up-regulated in JS during the early phases of cold stress. This study also identified some DEPs since they were up-regulated in JS or down-regulated in GE in terms of molecular mechanisms, which were mainly related to carbohydrate and energy metabolism, structure proteins, ROS scavening, amino acid metabolic and signaling transduction. Moreover, iTRAQ analysis was confirmed to be reliable via the results of phenotypes assays, metabolic analysis and q-PCR validation. Conclusion: By proteomics information,we found that the prompt response and the significant mobilization of proteins in JS maintained a higher level of cold tolerance, and the delay of cold response in GE could be a critical reason for the severe chilling injury. The candidate proteins we found will be the basis of future studies for further investigations and our findings may help to better understand the novel mechanisms of cold tolerance in cantaloupe.

scholarly journals EPEN-21. IMPAIRED NEURONAL-GLIAL FATE SPECIFICATION IN PEDIATRIC EPENDYMOMA REVEALED BY SINGLE-CELL RNA-SEQ

2020 ◽  
Vol 22 (Supplement_3) ◽  
pp. iii311-iii312
Author(s):  
Bernhard Englinger ◽  
Johannes Gojo ◽  
Li Jiang ◽  
Jens M Hübner ◽  
McKenzie L Shaw ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Single Cell ◽  
Regulatory Networks ◽  
Target Genes ◽  
Target Identification ◽  
Rna Seq ◽  
Cell Models ◽  
Pediatric Ependymoma ◽  
Glial Fate ◽  
Anatomic Locations

Abstract Ependymoma represents a heterogeneous disease affecting the entire neuraxis. Extensive molecular profiling efforts have identified molecular ependymoma subgroups based on DNA methylation. However, the intratumoral heterogeneity and developmental origins of these groups are only partially understood, and effective treatments are still lacking for about 50% of patients with high-risk tumors. We interrogated the cellular architecture of ependymoma using single cell/nucleus RNA-sequencing to analyze 24 tumor specimens across major molecular subgroups and anatomic locations. We additionally analyzed ten patient-derived ependymoma cell models and two patient-derived xenografts (PDXs). Interestingly, we identified an analogous cellular hierarchy across all ependymoma groups, originating from undifferentiated neural stem cell-like populations towards different degrees of impaired differentiation states comprising neuronal precursor-like, astro-glial-like, and ependymal-like tumor cells. While prognostically favorable ependymoma groups predominantly harbored differentiated cell populations, aggressive groups were enriched for undifferentiated subpopulations. Projection of transcriptomic signatures onto an independent bulk RNA-seq cohort stratified patient survival even within known molecular groups, thus refining the prognostic power of DNA methylation-based profiling. Furthermore, we identified novel potentially druggable targets including IGF- and FGF-signaling within poorly prognostic transcriptional programs. Ependymoma-derived cell models/PDXs widely recapitulated the transcriptional programs identified within fresh tumors and are leveraged to validate identified target genes in functional follow-up analyses. Taken together, our analyses reveal a developmental hierarchy and transcriptomic context underlying the biologically and clinically distinct behavior of ependymoma groups. The newly characterized cellular states and underlying regulatory networks could serve as basis for future therapeutic target identification and reveal biomarkers for clinical trials.

scholarly journals Identification of cold stress responsive microRNAs in cold tolerant and susceptible Hemerocallis fulva by high throughput sequencing

2020 ◽  
Author(s):  
Changbing Huang ◽  
Chun Jiang ◽  
limin Jin ◽  
Huanchao Zhang
Keyword(s):  
Low Temperature ◽  
Cold Stress ◽  
Cold Tolerance ◽  
Temperature Stress ◽  
Stress Responses ◽  
Target Genes ◽  
Low Temperature Stress ◽  
Differentially Expressed ◽  
Differentially Expressed Mirnas ◽  
Cold Tolerant

Abstract Background:Hemerocallis fulva is a perennial herb belonging to Hemerocallis of Hemerocallis. Because of the large and bright colors, it is often used as a garden ornamental plant. But most varieties of H. fulva on the market will wither in winter, which will affect their beauty. It is very important to study the effect of low temperature stress on the physiological indexes of H. fulva and understand the cold tolerance of different H. fulva. MiRNA is a kind of endogenous non coding small molecular RNA with length of 21-24nt. It mainly inhibits protein translation by cutting target genes, and plays an important role in the development of organisms, gene expression and biological stress. Low temperature is the main abiotic stress affecting the production of H. fulva in China, which hinders the growth and development of plants. A comprehensive understanding of the expression pattern of microRNA in H. fulva under low temperature stress can improve our understanding of microRNA mediated stress response. Although there are many studies on miRNAs of various plants under cold stress at home and abroad, there are few studies on miRNAs related to cold stress of H. fulva. It is of great significance to explore the cold stress resistant gene resources of H. fulva, especially the identification and functional research of miRNA closely related to cold stress, for the breeding of excellent H. fulva.Results A total of 5619 cold-responsive miRNAs, 315 putative novel and 5 304 conserved miRNAs, were identified from the leaves and roots of two different varieties ‘Jinyan’ (cold-tolerant) and ‘Lucretius ’ (cold-sensitive), which were stressed under -4 oC for 24 h. Twelve conserved and three novel miRNAs (novel-miR10, novel-miR19 and novel-miR48) were differentially expressed in leaves of ‘Jinyan’ under cold stress. Novel-miR19, novel-miR29 and novel-miR30 were up-regulated in roots of ‘Jinyan’ under cold stress. Thirteen and two conserved miRNAs were deferentially expressed in leaves and roots of ‘Lucretius’ after cold stress. The deferentially expressed miRNAs between two cultivars under cold stress include novel miRNAs and the members of the miR156, miR166 and miR319 families. A total of 6 598 target genes for 6 516 known miRNAs and 82 novel miRNAs were predicted by bioinformatic analysis, mainly involved in metabolic processes and stress responses. Ten differentially expressed miRNAs and predicted target genes were confirmed by quantitative reverse transcription PCR(q-PCR), and the expressional changes of target genes were negatively correlated to differentially expressed miRNAs. Our data indicated that some candidate miRNAs (e.g., miR156a-3-p, miR319a, and novel-miR19) may play important roles in plant response to cold stress.Conclusions Our study indicates that some putative target genes and miRNA mediated metabolic processes and stress responses are significant to cold tolerance in H. fulva.

scholarly journals Comparative Transcriptomics for Pepper (Capsicum annuum L.) under Cold Stress and after Rewarming

2021 ◽  
Vol 11 (21) ◽  
pp. 10204
Author(s):  
Wu Miao ◽  
Jingshuang Song ◽  
Yanwu Huang ◽  
Rongyun Liu ◽  
Gaofeng Zou ◽  
...  
Keyword(s):  
Transcription Factors ◽  
Cold Stress ◽  
Cold Tolerance ◽  
Inbred Line ◽  
Soluble Sugar ◽  
Cold Treatment ◽  
Nac Transcription Factors ◽  
Physiological Indicators ◽  
Damage Repair ◽  
Cytochrome P450 Family

Cold stress has become one of the main abiotic stresses in pepper, which severely limits the growth and development of pepper. In this study, the physiological indicators and transcriptome of a cold-tolerance (CT) inbred line A188 and a cold-sensitive (CS) inbred line A122 under cold–rewarm treatments were studied; the aim of this study was to determine the potential of the key factors in pepper response to cold stress. Compared with CT, CS wilts more seriously after cold stress, with poor resilience, higher content of malondialdehyde, and lower content of soluble sugar and total chlorophyll. Moreover, during cold treatment, 7333 and 5953 differentially expressed genes (DEGs) were observed for CT and CS, respectively. These DEGs were significantly enriched in pathways related to photosynthesis, plant hormone signal transduction, and DNA damage repair. Interestingly, in addition to the widely studied transcription factors related to cold, it was also found that 13 NAC transcription factors increased significantly in the T4 group; meanwhile, the NAC8 (Capana02g003557) and NAC72 (Capana07g002219) in CT were significantly higher than those in CS under rewarming for 1 h after 72 h cold treatment. Notably, weighted gene coexpression network analysis identified four positively correlated modules and eight hub genes, including zinc finger proteins, heat shock 70 kda protein, and cytochrome P450 family, which are related to cold tolerance. All of these pathways and genes may be responsible for the response to cold and even the cold tolerance in pepper.

scholarly journals ISMARA: Completely automated inference of gene regulatory networks from high-throughput data

2017 ◽  
Author(s):  
Mikhail Pachkov ◽  
Piotr J Balwierz ◽  
Phil Arnold ◽  
Andreas J Gruber ◽  
Mihaela Zavolan ◽  
...  
Keyword(s):  
Gene Expression ◽  
High Throughput ◽  
Regulatory Networks ◽  
Target Genes ◽  
Chromatin State ◽  
Response Analysis ◽  
Rna Seq ◽  
High Throughput Data ◽  
Micro Rnas ◽  
Gene Regulatory

As the costs of high-throughput measurement technologies continue to fall, experimental approaches in biomedicine are increasingly data intensive and the advent of big data is justifiably seen as holding the promise to transform medicine. However, as data volumes mount, researchers increasingly realize that extracting concrete, reliable, and actionable biological predictions from high-throughput data can be very challenging. Our laboratory has pioneered a number of methods for inferring key gene regulatory interactions from high-throughput data. For example, we developed motif activity response analysis (MARA)[, which models genome-wide gene expression (RNA-Seq, or microarray) and chromatin state (ChIP-Seq) data in terms of comprehensive predictions of regulatory sites for hundreds of mammalian regulators (TFs and micro-RNAs). Using these models, MARA identifies the key regulators driving gene expression and chromatin state changes, the activities of these regulators across the input samples, their target genes, and the sites on the genome through which these regulators act. We recently completely automated MARA in an integrated web-server (ismara.unibas.ch) that allows researchers to analyze their own data by simply uploading RNA-Seq or ChIP-Seq datasets, and provides results in an integrated web interface as well as in downloadable flat form.

scholarly journals Light Regulates the Cytokinin-Dependent Cold Stress Responses in Arabidopsis

2021 ◽  
Vol 11 ◽  
Author(s):  
Sylva Prerostova ◽  
Martin Černý ◽  
Petre I. Dobrev ◽  
Vaclav Motyka ◽  
Lucia Hluskova ◽  
...  
Keyword(s):  
Light Intensity ◽  
Cold Stress ◽  
Stress Tolerance ◽  
Stress Responses ◽  
Cold Treatment ◽  
Cytokinin Oxidase ◽  
Low Light ◽  
Cold Response ◽  
The Impact ◽  
Effect Of Light

To elucidate the effect of light intensity on the cold response (5°C; 7 days) in Arabidopsis thaliana, we compared the following parameters under standard light (150 μmol m–2 s–1), low light (20 μmol m–2 s–1), and dark conditions: membrane damage, photosynthetic parameters, cytokinin oxidase/dehydrogenase (CKX) activity, phytohormone levels, and transcription of selected stress- and hormone-related genes and proteome. The impact of cytokinins (CKs), hormones directly interacting with the light signaling pathway, on cold responses was evaluated using transformants overexpressing CK biosynthetic gene isopentenyl transferase (DEX:IPT) or CK degradation gene HvCKX2 (DEX:CKX) under a dexamethasone-inducible promoter. In wild-type plants, cold treatment under light conditions caused down-regulation of CKs (in shoots) and auxin, while abscisic acid (ABA), jasmonates, and salicylic acid (SA) were up-regulated, especially under low light. Cold treatment in the dark strongly suppressed all phytohormones, except ABA. DEX:IPT plants showed enhanced stress tolerance associated with elevated CK and SA levels in shoots and auxin in apices. Contrarily, DEX:CKX plants had weaker stress tolerance accompanied by lowered levels of CKs and auxins. Nevertheless, cold substantially diminished the impact from the inserted genes. Cold stress in dark minimized differences among the genotypes. Cold treatments in light strongly up-regulated stress marker genes RD29A, especially in roots, and CBF1-3 in shoots. Under control conditions, their levels were higher in DEX:CKX plants, but after 7-day stress, DEX:IPT plants exhibited the highest transcription. Transcription of genes related to CK metabolism and signaling showed a tendency to re-establish, at least partially, CK homeostasis in both transformants. Up-regulation of strigolactone-related genes in apices and leaves indicated their role in suppressing shoot growth. The analysis of leaf proteome revealed over 20,000 peptides, representing 3,800 proteins and 2,212 protein families (data available via ProteomeXchange, identifier PXD020480). Cold stress induced proteins involved in ABA and jasmonate metabolism, antioxidant enzymes, and enzymes of flavonoid and glucosinolate biosynthesis. DEX:IPT plants up-regulated phospholipase D and MAP-kinase 4. Cold stress response at the proteome level was similar in all genotypes under optimal light intensity, differing significantly under low light. The data characterized the decisive effect of light–CK cross-talk in the regulation of cold stress responses.

scholarly journals The Wheat GENIE3 Network Provides Biologically-Relevant Information in Polyploid Wheat

2020 ◽  
Vol 10 (10) ◽  
pp. 3675-3686 ◽  
Author(s):  
Sophie A. Harrington ◽  
Anna E. Backhaus ◽  
Ajit Singh ◽  
Keywan Hassani-Pak ◽  
Cristobal Uauy
Keyword(s):  
Transcription Factor ◽  
Transcription Factors ◽  
Candidate Gene ◽  
Web Application ◽  
Regulatory Networks ◽  
Target Genes ◽  
Gene Prediction ◽  
Relevant Information ◽  
Rna Seq ◽  
Biologically Relevant

Gene regulatory networks are powerful tools which facilitate hypothesis generation and candidate gene discovery. However, the extent to which the network predictions are biologically relevant is often unclear. Recently a GENIE3 network which predicted targets of wheat transcription factors was produced. Here we used an independent RNA-Seq dataset to test the predictions of the wheat GENIE3 network for the senescence-regulating transcription factor NAM-A1 (TraesCS6A02G108300). We re-analyzed the RNA-Seq data against the RefSeqv1.0 genome and identified a set of differentially expressed genes (DEGs) between the wild-type and nam-a1 mutant which recapitulated the known role of NAM-A1 in senescence and nutrient remobilisation. We found that the GENIE3-predicted target genes of NAM-A1 overlap significantly with the DEGs, more than would be expected by chance. Based on high levels of overlap between GENIE3-predicted target genes and the DEGs, we identified candidate senescence regulators. We then explored genome-wide trends in the network related to polyploidy and found that only homeologous transcription factors are likely to share predicted targets in common. However, homeologs which vary in expression levels across tissues are less likely to share predicted targets than those that do not, suggesting that they may be more likely to act in distinct pathways. This work demonstrates that the wheat GENIE3 network can provide biologically-relevant predictions of transcription factor targets, which can be used for candidate gene prediction and for global analyses of transcription factor function. The GENIE3 network has now been integrated into the KnetMiner web application, facilitating its use in future studies.

THE EFFECT OF HIGH TEMPERATURE STORAGE ON THE CAPACITY OF AN ICE-NUCLEATING-ACTIVE BACTERIUM AND FUNGUS TO REDUCE INSECT COLD-TOLERANCE

1995 ◽  
Vol 127 (1) ◽  
pp. 33-40 ◽  
Author(s):  
Paul Fields ◽  
Stéphan Pouleur ◽  
Claude Richard
Keyword(s):  
Cold Tolerance ◽  
Pseudomonas Syringae ◽  
Cold Hardiness ◽  
Insect Pest ◽  
Cold Treatment ◽  
Stored Grain ◽  
Supercooling Point ◽  
Ice Nuclei ◽  
The Difference ◽  
Cold Hardy

AbstractCold treatment is used to control the rusty grain beetle (Cryptolestes ferrugineus) (Coleoptera: Cucujidae), the predominant insect pest of stored grain in Canada. However, because it is difficult to cool the grain enough to control C. ferrugineus quickly, we have examined ways to reduce the cold-tolerance of adult C. ferrugineus, the most cold-hardy stage. We compared the efficacy of two ice nucleators, Pseudomonas syringae and Fusarium avenaceum, to decrease cold-tolerance of this insect, as well as their thermal stability. Ice nuclei from the bacteria P. syringae raised C. ferrugineus supercooling point from −17 to −6 °C, and increased mortality at −9°C for 24 h from 11 to 100%. Pseudomonas syringae held at 30°C for 16 weeks showed only a slight decline in its ability to reduce C. ferrugineus cold-tolerance. The fungus F. avenaceum raised the supercooling point of C. ferrugineus from −17 to −9°C, but only increased the mortality at −9°C for 24 h from 10 to 33%. Wheat treated with F. avenaceum and held at 30°C for 4 weeks reduced the cold-hardiness of C. ferrugineus, but had no effect after 8 weeks at 30°C. One reason for the difference between the two nucleators is that P. syringae had approximately 1000 times more ice nuclei per gram than did F. avenaceum. These results suggest that P. syringae is stable enough to reduce C. ferrugineus cold-tolerance after several weeks on warm grain. We discuss possible ways to increase the ice-nucleating activity of F. avenaceum.

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