A time-resolved multi-omics atlas of transcriptional regulation in response to high-altitude hypoxia across whole-body tissues.

High-altitude hypoxia acclimatization requires whole-body physiological regulation in highland immigrants, but the underlying genetic mechanism has not been clarified. Here we use sheep as an animal model for low-to-high altitude translocation. We generate multi-omics data including whole-genome sequences, time-resolved bulk RNA-Seq, ATAC-Seq and single-cell RNA-Seq from multiple tissues as well as phenotypic data from 20 bio-indicators. We characterize transcriptional changes of all genes in each tissue, and examine multi-tissue temporal dynamics and transcriptional interactions among genes. Particularly, we identify critical functional genes regulating the short response to hypoxia in each tissue (e.g., PARG in the cerebellum and HMOX1 in the colon). We further identify TAD-constrained cis-regulatory elements, which suppress the transcriptional activity of most genes under hypoxia. Phenotypic and transcriptional evidence indicate that antenatal hypoxia could improve hypoxia tolerance in offspring. Furthermore, we provide time-series expression data of candidate genes associated with human mountain sickness (e.g., BMPR2) and high-altitude adaptation (e.g., HIF1A). Our study provides valuable resources and insights for future hypoxia-related studies in mammals.

Plasticity of Gene Expression and Thermal Tolerance: Implications for Climate Change Vulnerability in a Tropical Forest Lizard.

AbstractTropical ectotherms are thought to be especially vulnerable to climate change because they have evolved in temporally stable thermal environments and therefore have decreased tolerance for thermal variability. Thus, they are expected to have narrow thermal tolerance ranges, live close to their upper thermal tolerance limits, and have decreased thermal acclimation capacity. Although models often predict that tropical forest ectotherms are especially vulnerable to rapid environmental shifts, these models rarely include the potential for plasticity of relevant traits. We measured phenotypic plasticity of thermal tolerance and thermal preference as well as multitissue transcriptome plasticity in response to warmer temperatures in a species that previous work has suggested is highly vulnerable to climate warming, the Panamanian slender anole lizard (Anolis apletophallus). We found that many genes, including heat shock proteins, were differentially expressed across tissues in response to short-term warming. Under long-term warming, the voluntary thermal maxima of lizards also increased, although thermal preference exhibited only limited plasticity. Using these data, we modeled changes in the activity time of slender anoles through the end of the century under climate change and found that plasticity should delay declines in activity time by at least two decades. Our results suggest that slender anoles, and possibly other tropical ectotherms, can alter the expression of genes and phenotypes when responding to shifting environmental temperatures and that plasticity should be considered when predicting the future of organisms under a changing climate.

Integrated analysis of ATAC-seq and transcriptomic reveals the ScDof3-ScproC molecular module regulating the cold acclimation capacity of potato.

Low temperature severely affects the geographical distribution and production of potato, which may incur cold damage in early spring or winter. Cultivated potatoes, mainly derived from Solanum tuberosum, are sensitive to freezing stress, but wild species of potato such as S. commersonii exhibit both constitutive freezing tolerance and/or cold acclimation tolerance. Hence, such wild species could assist in cold hardiness breeding. Yet the key transcription factors and their downstream functional genes that confer freezing tolerance are far from clear, hindering the breeding process. Here, we used ATAC-seq (Assay for Transposase-Accessible Chromatin with high-throughput sequencing) alongside RNA-seq to investigate the variation in chromatin accessibility and patterns of gene expression in freezing-tolerant CMM5 (S. commersonii), before and after its cold treatment. Our results suggest that after exposure to cold, transcription factors including Dof3, ABF2, PIF4, and MYB4 were predicted to further control the genes active in the synthetic/metabolic pathways of plant hormones, namely abscisic acid, polyamine, and reductive glutathione (among others). This suggests these transcription factors could regulate freezing tolerance of CMM5 leaves. In particular, ScDof3 was proven to regulate the expression of ScproC (pyrroline-5-carboxylate reductase, P5CR) according to dual-LUC assays. Overexpressing ScDof3 in Nicotiana benthamiana leaves led to an increase in both the proline content and expression level of NbproC (homolog of ScproC). These results demonstrate the ScDof3-ScproC module regulates the proline content and thus promotes freezing tolerance in potato. Our research provides valuable genetic resources to further study the molecular mechanisms underpinning cold tolerance in potato.

Gene expression plasticity facilitates acclimatization of a long-lived Caribbean coral across divergent reef environments.

Local adaptation can increase fitness under stable environmental conditions. However, in rapidly changing environments, compensatory mechanisms enabled through plasticity may better promote fitness. Climate change is causing devastating impacts on coral reefs globally and understanding the potential for adaptive and plastic responses is critical for reef management. We conducted a four-year, three-way reciprocal transplant of the Caribbean coral Siderastrea siderea across forereef, backreef, and nearshore populations in Belize to investigate the potential for environmental specialization versus plasticity in this species. Corals maintained high survival within forereef and backreef environments, but transplantation to nearshore environments resulted in high mortality, suggesting that nearshore environments present strong environmental selection. Only forereef-sourced corals demonstrated evidence of environmental specialization, exhibiting the highest growth in the forereef. Gene expression profiling 3.5 years post-transplantation revealed that transplanted coral hosts exhibited profiles more similar to other corals in the same reef environment, regardless of their source location, suggesting that transcriptome plasticity facilitates acclimatization to environmental change in S. siderea. In contrast, algal symbiont (Cladocopium goreaui) gene expression showcased functional variation between source locations that was maintained post-transplantation. Our findings suggest limited acclimatory capacity of some S. siderea populations under strong environmental selection and highlight the potential limits of coral physiological plasticity in reef restoration.

TaMAPK3 phosphorylates TaCBF and TaICE and plays a negative role in wheat freezing tolerance.

Freezing temperature during overwintering often kills plants; plants have thus, developed a defense mechanism called 'cold acclimation', in which a number of genes are involved in increasing cell protection and gene expression. Mitogen-activated protein kinase (MAPK) controls proteins' activities by phosphorylation and is involved in numerous metabolic pathways. In this study, we identified the protein interaction between TaMAPK3 and the proteins in the cold response pathway, ICE41, ICE87, and CBFIVd-D9. The subcellular localization and bimolecular fluorescence complement (BiFC) assays revealed that these proteins interact in the nucleus or in the plasma membrane. Furthermore, MAPK3-mediated phosphorylation of ICE41, ICE87, and CBFIVd-D9 was verified using an in vitro phosphorylation assay. TaMAPK3-overexpressing transgenic Brachypodium showed a lower survival rate upon freezing stress and lower proline content during cold acclimation, compared to wild-type plants. Furthermore, cold response gene expression analysis revealed that the expression of these genes was suppressed in the transgenic lines under cold treatment. It was further elucidated that MAPK3 mediates the degradation of ICE and CBF proteins, which implies the negative impact of MAPK3 on the freezing tolerance of plants. This study will help to elucidate the molecular mechanisms of cold tolerance and the activity of MAPK3 in wheat.

Temperate woody species across the angiosperm phylogeny acquire tolerance to water deficit stress during the growing season.

Understanding the capacity of temperate trees to acclimate to limited soil water has become essential in the face of increasing drought risk due to climate change. We documented seasonal - or phenological - patterns in acclimation to water deficit stress in stems and leaves of tree species spanning the angiosperm phylogeny. Over 3 yr of field observations carried out in two US arboreta, we measured stem vulnerability to embolism (36 individuals of 7 Species) and turgor loss point (119 individuals of 27 species) over the growing season. We also conducted a growth chamber experiment on 20 individuals of one species to assess the mechanistic relationship between soil water restriction and acclimation. In three-quarters of species measured, plants became less vulnerable to embolism and/or loss of turgor over the growing season. We were able to stimulate this acclimatory effect by withholding water in the growth chamber experiment. Temperate angiosperms are capable of acclimation to soil water deficit stress, showing maximum vulnerability to soil water deficits following budbreak and becoming more resilient to damage over the course of the growing season or in response to simulated drought. The species-specific tempo and extent of this acclimatory potential constitutes preadaptive climate change resilience.

Multiomics analyses reveal the central role of the nucleolus and its machinery during heat stress acclimation in Pinus radiata.

Global warming is causing rapid changes in mean annual temperature and more severe drought periods. These are major contributors of forest dieback, which is becoming more frequent and widespread. In this work, we investigated how the transcriptome of Pinus radiata changed during initial heat stress response and acclimation. To this end, we generated a high-density dataset employing Illumina technology. This approach allowed us to reconstruct a needle transcriptome, defining 12 164 and 13 590 transcripts as down- and up-regulated, respectively, during a time course stress acclimation experiment. Additionally, the combination of transcriptome data with other available omics layers allowed us to determine the complex inter-related processes involved in the heat stress response from the molecular to the physiological level. Nucleolus and nucleoid activities seem to be a central core in the acclimating process, producing specific RNA isoforms and other essential elements for anterograde-retrograde stress signaling such as NAC proteins (Pra_vml_051671_1 and Pra_vml_055001_5) or helicase RVB. These mechanisms are connected by elements already known in heat stress response (redox, heat-shock proteins, or abscisic acid-related) and with others whose involvement is not so well defined such as shikimate-related, brassinosteriods, or proline proteases together with their potential regulatory elements. This work provides a first in-depth overview about molecular mechanisms underlying the heat stress response and acclimation in P. radiata.

Differential responses to UCP1 ablation in classical brown versus beige fat, despite a parallel increase in sympathetic innervation.

In the cold, the absence of the mitochondrial uncoupling protein 1 (UCP1) results in hyper-recruitment of beige fat, but classical brown fat becomes atrophied. Here we examine possible mechanisms underlying this phenomenon. We confirm that in brown fat from UCP1-knockout (UCP1-KO) mice acclimated to the cold, the levels of mitochondrial respiratory chain proteins were diminished; however, in beige fat, the mitochondria seemed to be unaffected. The macrophages that accumulated massively not only in brown fat but also in beige fat of the UCP1-KO mice acclimated to cold did not express tyrosine hydroxylase, the norepinephrine transporter (NET) and monoamine oxidase-A (MAO-A). Consequently, they could not influence the tissues through the synthesis or degradation of norepinephrine. Unexpectedly, in the cold, both brown and beige adipocytes from UCP1-KO mice acquired an ability to express MAO-A. Adipose tissue norepinephrine was exclusively of sympathetic origin, and sympathetic innervation significantly increased in both tissues of UCP1-KO mice. Importantly, the magnitude of sympathetic innervation and the expression levels of genes induced by adrenergic stimulation were much higher in brown fat. Therefore, we conclude that no qualitative differences in innervation or macrophage character could explain the contrasting reactions of brown versus beige adipose tissues to UCP1-ablation. Instead, these contrasting responses may be explained by quantitative differences in sympathetic innervation: the beige adipose depot from the UCP1-KO mice responded to cold acclimation in a canonical manner and displayed enhanced recruitment, while the atrophy of brown fat lacking UCP1 may be seen as a consequence of supraphysiological adrenergic stimulation in this tissue.

G protein-coupled receptor-based thermosensation determines temperature acclimatization of Caenorhabditis elegans.

Animals must sense and acclimatize to environmental temperatures for survival, yet their thermosensing mechanisms other than transient receptor potential (TRP) channels remain poorly understood. We identify a trimeric G protein-coupled receptor (GPCR), SRH-40, which confers thermosensitivity in sensory neurons regulating temperature acclimatization in Caenorhabditis elegans. Systematic knockdown of 1000 GPCRs by RNAi reveals GPCRs involved in temperature acclimatization, among which srh-40 is highly expressed in the ADL sensory neuron, a temperature-responsive chemosensory neuron, where TRP channels act as accessorial thermoreceptors. In vivo Ca2+ imaging demonstrates that an srh-40 mutation reduced the temperature sensitivity of ADL, resulting in supranormal temperature acclimatization. Ectopically expressing SRH-40 in a non-warmth-sensing gustatory neuron confers temperature responses. Moreover, temperature-dependent SRH-40 activation is reconstituted in Drosophila S2R+ cells. Overall, SRH-40 may be involved in thermosensory signaling underlying temperature acclimatization. We propose a dual thermosensing machinery through a GPCR and TRP channels in a single sensory neuron.

Trehalose 6-phosphate synthase gene rdtps1 contributes to thermal acclimation in Rhyzopertha dominica.

BACKGROUND: The lesser grain borer (Rhyzopertha dominica), a worldwide primary pest of stored grain, causes serious economic losses and threatens stored food safety. R. dominica can respond to changes in temperature, especially the adaptability to heat. In this study, transcriptome analysis of R. dominica exposed to different temperatures was performed to elucidate differences in gene expression and the underling molecular mechanism. RESULTS: Isoform-sequencing generated 17,721,200 raw reads and yielded 20,416 full-length transcripts. A total of 18,880 (92.48%) transcripts were annotated. We extracted RNA from R. dominica reared at 5 °C (cold stress), 15 °C (cold stress), 27 °C (ambient temperature) and 40 °C (heat stress) for RNA-seq. Compared to those of control insects reared at 27 °C, 119, 342, and 875 differentially expressed genes (DEGs) were identified at 5 °C, 15 °C, and 40 °C, respectively. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis revealed that pathways associated with "fatty acid metabolism", "fatty acid biosynthesis", "AMPK signaling pathway", "neuroactive ligand receptor interaction", and "longevity regulating pathway-multiple species" were significantly enriched. The functional annotation revealed that the genes encoding heat shock proteins (HSPs), fatty acid synthase (FAS), phospholipases (PLA), trehalose transporter (TPST), trehalose 6-phosphate synthase (TPS), and vitellogenin (Vg) were most likely involved in temperature regulation, which was also validated by RT-qPCR. Seven candidate genes (rdhsp1, rdfas1, rdpla1, rdtpst1, rdtps1, rdvg1, and rdP450) were silenced in the RNA interference (RNAi) assay. RNAi of each candidate gene suggested that inhibiting rdtps1 expression significantly decreased the trehalose level and survival rate of R. dominica at 40 °C. CONCLUSIONS: These results indicated that trehalose contributes to the high temperature resistance of R. dominica. Our study elucidates the molecular mechanisms underlying heat tolerance and provides a potential target for the pest management in R. dominica.

Natural genetic variation in GLK1-mediated photosynthetic acclimation in response to light.

BACKGROUND: GOLDEN-like (GLK) transcription factors are central regulators of chloroplast biogenesis in Arabidopsis and other species. Findings from Arabidopsis show that these factors also contribute to photosynthetic acclimation, e.g. to variation in light intensity, and are controlled by retrograde signals emanating from the chloroplast. However, the natural variation of GLK1-centered gene-regulatory networks in Arabidopsis is largely unexplored. RESULTS: By evaluating the activities of GLK1 target genes and GLK1 itself in vegetative leaves of natural Arabidopsis accessions grown under standard conditions, we uncovered variation in the activity of GLK1 centered regulatory networks. This is linked with the ecogeographic origin of the accessions, and can be associated with a complex genetic variation across loci acting in different functional pathways, including photosynthesis, ROS and brassinosteroid pathways. Our results identify candidate upstream regulators that contribute to a basal level of GLK1 activity in rosette leaves, which can then impact the capacity to acclimate to different environmental conditions. Indeed, accessions with higher GLK1 activity, arising from habitats with a high monthly variation in solar radiation levels, may show lower levels of photoinhibition at higher light intensities. CONCLUSIONS: Our results provide evidence for natural variation in GLK1 regulatory activities in vegetative leaves. This variation is associated with ecogeographic origin and can contribute to acclimation to high light conditions.

Structural changes in cell wall pectic polymers contribute to freezing tolerance induced by cold acclimation in plants.

Subzero temperatures are often lethal to plants. Many temperate herbaceous plants have a cold acclimation mechanism that allows them to sense a drop in temperature and prepare for freezing stress through accumulation of soluble sugars and cryoprotective proteins. As ice formation primarily occurs in the apoplast (the cell wall space), cell wall functional properties are important for plant freezing tolerance. Although previous studies have shown that the amounts of constituent sugars of the cell wall, in particular those of pectic polysaccharides, are altered by cold acclimation, the significance of this change during cold acclimation has not been clarified. We found that ß-1,4-galactan, which forms neutral side chains of the acidic pectic rhamnogalacturonan-I, accumulates in the cell walls of Arabidopsis and various freezing-tolerant vegetables during cold acclimation. The gals1 gals2 gals3 triple mutant, which has reduced ß-1,4-galactan in the cell wall, exhibited impaired freezing tolerance compared with wild-type Arabidopsis during initial stages of cold acclimation. Expression of genes involved in the galactan biosynthesis pathway, such as galactan synthases and UDP-glucose 4-epimerases, was induced during cold acclimation in Arabidopsis, explaining the galactan accumulation. Cold acclimation resulted in a decrease in extensibility and an increase in rigidity of the cell wall in the wild type, whereas these changes were not observed in the gals1 gals2 gals3 triple mutant. These results indicate that the accumulation of pectic ß-1,4-galactan contributes to acquired freezing tolerance by cold acclimation, likely via changes in cell wall mechanical properties.

Mixed Patterns of Intergenerational DNA Methylation Inheritance in Acropora.

For sessile organisms at high risk from climate change, phenotypic plasticity can be critical to rapid acclimation. Epigenetic markers like DNA methylation are hypothesized as mediators of plasticity; methylation is associated with the regulation of gene expression, can change in response to ecological cues, and is a proposed basis for the inheritance of acquired traits. Within reef-building corals, gene-body methylation (gbM) can change in response to ecological stressors. If coral DNA methylation is transmissible across generations, this could potentially facilitate rapid acclimation to environmental change. We investigated methylation heritability in Acropora, a stony reef-building coral. Two Acropora millepora and two Acropora selago adults were crossed, producing eight offspring crosses (four hybrid, two of each species). We used whole-genome bisulfite sequencing to identify methylated loci and allele-specific alignments to quantify per-locus inheritance. If methylation is heritable, differential methylation (DM) between the parents should equal DM between paired offspring alleles at a given locus. We found a mixture of heritable and nonheritable loci, with heritable portions ranging from 44% to 90% among crosses. gBM was more heritable than intergenic methylation, and most loci had a consistent degree of heritability between crosses (i.e. the deviation between parental and offspring DM were of similar magnitude and direction). Our results provide evidence that coral methylation can be inherited but that heritability is heterogenous throughout the genome. Future investigations into this heterogeneity and its phenotypic implications will be important to understanding the potential capability of intergenerational environmental acclimation in reef building corals.

A large-effect fitness trade-off across environments is explained by a single mutation affecting cold acclimation.

Identifying the genetic basis of local adaptation and fitness trade-offs across environments is a central goal of evolutionary biology. Cold acclimation is an adaptive plastic response for surviving seasonal freezing, and costs of acclimation may be a general mechanism for fitness trade-offs across environments in temperate zone species. Starting with locally adapted ecotypes of Arabidopsis thaliana from Italy and Sweden, we examined the fitness consequences of a naturally occurring functional polymorphism in CBF2. This gene encodes a transcription factor that is a major regulator of cold-acclimated freezing tolerance and resides within a locus responsible for a genetic trade-off for long-term mean fitness. We estimated the consequences of alternate genotypes of CBF2 on 5-y mean fitness and fitness components at the native field sites by comparing near-isogenic lines with alternate genotypes of CBF2 to their genetic background ecotypes. The effects of CBF2 were validated at the nucleotide level using gene-edited lines in the native genetic backgrounds grown in simulated parental environments. The foreign CBF2 genotype in the local genetic background reduced long-term mean fitness in Sweden by more than 10%, primarily via effects on survival. In Italy, fitness was reduced by more than 20%, primarily via effects on fecundity. At both sites, the effects were temporally variable and much stronger in some years. The gene-edited lines confirmed that CBF2 encodes the causal variant underlying this genetic trade-off. Additionally, we demonstrated a substantial fitness cost of cold acclimation, which has broad implications for potential maladaptive responses to climate change.

Metabolic and immune costs balance during natural acclimation of corals in fluctuating environments.

Epigenetic modifications based on DNA methylation can rapidly improve the potential of corals to adapt to environmental pressures by increasing their phenotypic plasticity, a factor important for scleractinian corals to adapt to future global warming. However, the extent to which corals develop similar adaptive mechanisms and their specific adaptation processes remain unclear. Here, to reveal the regulatory mechanism by which DNA methylation improves thermal tolerance in Pocillopora damicornis under fluctuating environments, we analyzed genome-wide DNA methylation signatures in P. damicornis and compared the differences in the methylation and transcriptional responses of P. damicornis from fluctuating and stable environments using whole-genome bisulfite sequencing and nanopore-based RNA sequencingtranscriptome sequencing. We discovered low methylation levels in P. damicornis (average methylation 4.14%), with CpG accounting for 74.88%, CHH for 13.27%, and CHG for 11.85% of this methylation. However, methylation levels did not change between coral samples from the fluctuating and stable environments. The varied methylation levels in different regions of the gene revealed that the overall methylation level of the gene body was relatively high and showed a bimodal methylation pattern. Methylation occurs primarily in exons rather than introns within the gene body In P. damicornis, there was only a weak correlation between methylation and transcriptional changes at the individual gene level, and the methylation and gene expression levels generally exhibited a bell-shaped relationship, which we speculate may be due to the specificity of cnidarian species. Correlation analysis between methylation levels and the transcriptome revealed that the highest proportion of the top 20 enriched KEGG pathways was related to immunity. Additionally, P. damicornis collected from a high-temperature pool had a lower metabolic rate than those collected from a low-temperature pool. We hypothesize that the dynamic balance of energy-expenditure costs between immunity and metabolism is an important strategy for increasing P. damicornis tolerance. The fluctuating environment of high-temperature pools may increase the heat tolerance in corals by increasing their immunity and thus lowering their metabolism.

MSTRG3207 promotes apoptosis in zebrafish ZF4 cells via sponging dre-miR-736/bbc3/LOC101885512 axis during cold acclimation.

Long non-coding RNAs (lncRNAs) play essential roles in a variety of biological processes. It has been recently reported that lncRNAs can regulate mRNA expression by binding to microRNAs (miRNAs) as competing endogenous RNAs (ceRNAs). However, the involvement of this regulatory mechanism during cold acclimation in fish remains unclear. In this study, we constructed a ceRNA network mediated by lncRNAs in cold-acclimated zebrafish ZF4 cells through bioinformatic analysis of the mRNA, miRNA, and lncRNA profiles obtained from ZF4 cells cultured at 18 °C for 30 days. A previously uncharacterized lncRNA, MSTRG3207, was selected for further analysis. MSTRG3207 was upregulated and dre-miR-736 was downregulated during cold acclimation. MSTRG3207 was cloned by rapid amplification of cDNA ends (RACE) and functionally characterized. The binding of MSTRG3207 to dre-miR-736 was validated by dual-luciferase reporter assay. Under cold acclimation, MSTRG3207 promoted apoptosis by sponging dre-miR-736 and upregulating bbc3 and LOC101885512, two apoptotic genes targeted by dre-miR-736. Taken together, our findings indicate that MSTRG3207 upregulation promotes apoptosis by sponging dre-miR-736 during cold acclimation in fish.

Phenotypic plasticity for improved light harvesting, in tandem with methylome repatterning in reef-building corals.

Acclimatization through phenotypic plasticity represents a more rapid response to environmental change than adaptation and is vital to optimize organisms' performance in different conditions. Generally, animals are less phenotypically plastic than plants, but reef-building corals exhibit plant-like properties. They are light dependent with a sessile and modular construction that facilitates rapid morphological changes within their lifetime. We induced phenotypic changes by altering light exposure in a reciprocal transplant experiment and found that coral plasticity is a colony trait emerging from comprehensive morphological and physiological changes within the colony. Plasticity in skeletal features optimized coral light harvesting and utilization and paralleled significant methylome and transcriptome modifications. Network-associated responses resulted in the identification of hub genes and clusters associated to the change in phenotype: inter-partner recognition and phagocytosis, soft tissue growth and biomineralization. Furthermore, we identified hub genes putatively involved in animal photoreception-phototransduction. These findings fundamentally advance our understanding of how reef-building corals repattern the methylome and adjust a phenotype, revealing an important role of light sensing by the coral animal to optimize photosynthetic performance of the symbionts.

Integrative analysis of physiology and genomics provides insights into freeze tolerance adaptations of Acacia koa along an elevational cline.

Natural selection for plant species in heterogeneous environments creates genetic variation for traits such as cold tolerance. While physiological or molecular analyses have been used to evaluate stress tolerance adaptations, combining these approaches may provide deeper insight. Acacia koa (koa) occurs from sea level to 2300 m in Hawai'i, USA. At high elevations, natural koa populations have declined due to deforestation, and freeze tolerance is a limiting factor for tree regeneration. We used physiology and molecular analyses to evaluate cold tolerance of koa populations from low (300-750 m), middle (750-1500 m), and high elevations (1500-2100 m). Half of the seedlings were cold acclimated by exposure to progressively lowered air temperatures for eight weeks (from 25.6/22.2°C to 8/4°C, day/night). Using the whole plant physiology-freezing test and koa C-repeat Binding Factor CBF genes, our results indicated that koa can be cold-acclimated when exposed to low, non-freezing temperatures. Seedlings from high elevations had consistently higher expression of Koa CBF genes associated with cold tolerance, helping to explain variation in cold-hardy phenotypes. Evaluation of the genetic background of 22 koa families across the elevations with low coverage RNA sequencing indicated that high elevation koa had relatively low values of heterozygosity, suggesting that adaptation is more likely to arise in the middle and low elevation sources. This physiology and molecular data for cold tolerance of koa across the elevation gradient of the Hawaiian Islands provides insights into natural selection processes and may help to support guidelines for conservation and seed transfer in forest restoration efforts.