Phenotyping 172 strawberry genotypes for water soaking reveals a close relationship with skin water permeance.

Water soaking is a commercially important disorder of field-grown strawberries that is exacerbated by surface wetness and high humidity. The objective was to establish the effect of genotype on susceptibility to water soaking. Three greenhouse-grown model 'collections' were used comprising a total of 172 different genotypes: (1) a segregating F2 population, (2) a collection of strawberry cultivars and breeding clones, and (3) a collection of wild Fragaria species. A standardized immersion assay was used to induce water soaking. Potential relationships between water soaking and water uptake characteristics, depth of the achene depressions, fruit firmness, cuticle mass and strain relaxation and microcracking were investigated. Further, the effect of downregulating the polygalacturonase genes (FaPG1 and FaPG2) on the susceptibility to water soaking was investigated. The collection of wild species was most susceptible to water soaking. This was followed by the collection of cultivars and breeding clones, and by the F2 population. Susceptibility to water soaking was strongly correlated with water uptake rate (mass of water, per fruit, per time). For the pooled dataset of 172 genotypes, 46% of the variability in water soaking was accounted for by the permeance of the skin to osmotic water uptake. Susceptibility to water soaking was not, or was only poorly correlated with measurements of fruit surface area or of the osmotic potential of the expressed fruit juice. The only exceptions were the wild Fragaria species which were highly variable in fruit size and also in fruit osmotic potential. For genotypes from the F2 and the wild species collections, firmer fruit were less susceptible to water soaking than softer fruit. There were no relationships between fruit firmness and susceptibility to water soaking in transgenic plants in which FaPG1 and FaPG2 were down-regulated. Susceptibility to water soaking was not related to cuticle mass per unit fruit surface area, nor to strain relaxation of the cuticle upon isolation, nor to achene position. In summary, strawberry's susceptibility to water soaking has a significant genetic component and is closely and consistently related to the skin's permeance to osmotic water uptake.

Glycosylphosphatidylinositol-anchored lipid transfer proteins influence root cap cuticle formation at primary root tips, promoting NaCl tolerance in Arabidopsis seedlings.

Root cap cuticles (RCCs), comprising mainly very-long-chain fatty acids (VLCFAs), promote salt tolerance by preventing ion influx. Glycosylphosphatidylinositol-anchored lipid transfer protein (LTPG)1 and LTPG2 participate in VLCFA deposition in the extracellular region, aiding RCC formation in the lateral roots. In this study, we investigated whether LTPG1 and LTPG2 have similar functions in the primary roots of young Arabidopsis thaliana. Phenotypic analyses, fluorescence microscopy, and RT-qPCR confirmed that NaCl exposure induced LTPG1 and LTPG2 expression and promoted RCC formation in young primary roots. The loss of RCC in the ltpg1 and ltpg2 mutants resulted in increased NaCl sensitivity of root elongation. NaCl also upregulated the expression of several NaCl-responsive genes in ltpg1 and ltpg2. We conclude that RCC formation via LTPG function is pivotal in enhancing salt tolerance in young primary roots.

The beetle's structural protein CPCFC making elytra tough and rigid.

The insect cuticle, which serves as both a protective barrier and an efficient lever system for locomotion, is an extracellular matrix primarily composed of chitin and protein. The cuticle protein CPCFC characterized by a "CFC" motif containing 2 Cys split by the insertion of 5 residues is distributed across most insect species and specifically localized in the hard part of the cuticle. However, their physiological function is not fully understood. Here, we report 2 CPCFC proteins, TcCPCFC1 and TcCPCFC2, derived from the Coleopteran insect Tribolium castaneum. We revealed that TcCPCFC1 and TcCPCFC2 were predominantly expressed during the larval and adult stages of T. castaneum, respectively. The transcription downregulation of TcCPCFC1 significantly decreased the modulus and toughness of the elytral cuticle. We found that TcCPCFC proteins have high binding affinity to chitin. We cloned and produced recombinant TcCPCFC proteins and demonstrated that the addition of TcCPCFC proteins to chitin hydrogel greatly enhanced the hydrogel's modulus and toughness by forming denser chitin fibrous networks. Our findings reveal the functional role of CPCFC proteins in enhancing mechanical properties of insect cuticle, and we validate this process in vitro, and offer a protein candidate for fabrication of advanced chitin-based materials.

An advanced metabolomic approach on grape skins untangles cultivar preferences by Drosophila suzukii for oviposition.

Insects' host preferences are regulated by multiple factors whose interactions are only partly understood. Here we make use of an in-depth, untargeted metabolomic approach combining molecular networking (MN) and supervised Analysis of variance Multiblock Orthogonal Partial Least Squares (AMOPLS) to untangle egg-laying preferences of Drosophila suzukii, an invasive, highly polyphagous and destructive fruit pest originating from Southeast Asia. Based on behavioural experiments in the laboratory as well as field observation, we selected eight genetically related Vitis vinifera cultivars (e.g., Ancellotta, Galotta, Gamaret, Gamay, Gamay précoce, Garanoir, Mara and Reichensteiner) exhibiting significant differences in their susceptibility toward D. suzukii. The two most and the two least attractive red cultivars were chosen for further metabolomic analyses of their grape skins. The combination of MN and statistical AMOPLS findings with semi-quantitative detection information enabled us to identify flavonoids as interesting markers for differences in the attractiveness of the four studied grape cultivars towards D. suzukii. Overall, dihydroflavonols were accumulated in unattractive grape cultivars, while attractive grape cultivars were richer in flavonols. Crucially, both dihydroflavonols and flavonols were abundant metabolites in the semi-quantitative analysis of the extracted molecules from the grape skin. We discuss how these two flavonoid classes might influence the egg-laying behaviour of D. suzukii females and how they could serve as potential markers for D. suzukii infestations in grapes that can be potentially extended to other fruits. We believe that our novel, integrated analytical approach could also be applied to the study of other biological relationships characterised by multiple evolving parameters.

Genome-Wide Association Study of Cuticle and Lipid Droplet Properties of Cucumber (Cucumis sativus L.) Fruit.

The fruit surface is a critical first line of defense against environmental stress. Overlaying the fruit epidermis is the cuticle, comprising a matrix of cutin monomers and waxes that provides protection and mechanical support throughout development. The epidermal layer of the cucumber (Cucumis sativus L.) fruit also contains prominent lipid droplets, which have recently been recognized as dynamic organelles involved in lipid storage and metabolism, stress response, and the accumulation of specialized metabolites. Our objective was to genetically characterize natural variations for traits associated with the cuticle and lipid droplets in cucumber fruit. Phenotypic characterization and genome-wide association studies (GWAS) were performed using a resequenced cucumber core collection accounting for >96% of the allelic diversity present in the U.S. National Plant Germplasm System collection. The collection was grown in the field, and fruit were harvested at 16-20 days post-anthesis, an age when the cuticle thickness and the number and size of lipid droplets have stabilized. Fresh fruit tissue sections were prepared to measure cuticle thickness and lipid droplet size and number. The collection showed extensive variation for the measured traits. GWAS identified several QTLs corresponding with genes previously implicated in cuticle or lipid biosynthesis, including the transcription factor SHINE1/WIN1, as well as suggesting new candidate genes, including a potential lipid-transfer domain containing protein found in association with isolated lipid droplets.

Improving Cuticle Thickness and Quality Traits in Table Grape cv. 'Italia' Using Pre-Harvest Treatments.

Table grape viticulture, due to the impact of climate change, will have to face many challenges in the coming decades, including resistance to pathogens and physiological disorders. Our attention was focused on fruit cracking due to its ubiquitous presence in several species. This study explores the effects of three different treatments on the epidermis and cuticle of table grape berries by evaluating the impact of the girdling technique on various fruit quality parameters, including cuticle thickness, sugar content, acidity, color, bunch weight, and rheological properties. The treatments were (1) calcium chloride (CaCl2), (2) calcium chloride + salicylic acid (CaCl2 + SA), and (3) calcium chloride + Ascophyllum nodosum (CaCl2 + AN), with and without girdling, plus an untreated control. This research was conducted over the 2021-2022 growing season in a commercial vineyard in Licodia Eubea, Sicily, Italy. The results indicate significant variations in cuticle thickness and other qualitative traits throughout the growth and ripening phases, with notable differences depending on the treatment used. This study's findings suggest that specific treatments can influence the structural integrity of the grape cuticle, potentially impacting the fruit's susceptibility to cracking and overall marketability. The findings provide valuable insights into the role of chemical treatments and cultural techniques in enhancing fruit quality and resistance to environmental stresses in table grape cultivation.

The evolution of plant responses underlying specialized metabolism in host-pathogen interactions.

In the course of plant evolution from aquatic to terrestrial environments, land plants (embryophytes) acquired a diverse array of specialized metabolites, including phenylpropanoids, flavonoids and cuticle components, enabling adaptation to various environmental stresses. While embryophytes and their closest algal relatives share candidate enzymes responsible for producing some of these compounds, the complete genetic network for their biosynthesis emerged in embryophytes. In this review, we analysed genomic data from chlorophytes, charophytes and embryophytes to identify genes related to phenylpropanoid, flavonoid and cuticle biosynthesis. By integrating published research, transcriptomic data and metabolite studies, we provide a comprehensive overview on how these specialized metabolic pathways have contributed to plant defence responses to pathogens in non-vascular bryophytes and vascular plants throughout evolution. The evidence suggests that these biosynthetic pathways have provided land plants with a repertoire of conserved and lineage-specific compounds, which have shaped immunity against invading pathogens. The discovery of additional enzymes and metabolites involved in bryophyte responses to pathogen infection will provide evolutionary insights into these versatile pathways and their impact on environmental terrestrial challenges.This article is part of the theme issue 'The evolution of plant metabolism'.

Nature's loom: How to design a spinning tool using chitin-protein based composite material.

Silk-producing animals use spigots to generate natural silk fibers for various purposes. These natural looms must be able to withstand prolonged silk extrusion. To gain insight into the functional basis of spigots, we report on the structural design of the spigot of the silkworm Bombyx mori. The B. mori spigot exhibits a unique triple-ridged strip surface structure, consisting of cuticle proteins, resilin, chitin, and metal ions (such as K and Ca). This multi-microstructure endows the spigot with superior hierarchical mechanical properties, enabling it to function as a spinning tool for silk formation, thereby influencing the structure and performance of the silk. These findings demonstrate new pathways for achieving specialized functions in confined spaces, providing theoretical support for understanding the natural spinning mechanism and inspiring new directions for developing innovative biomimetic materials.

Compositional variances in petal cuticular wax of eight rose species and their impacts on vase life under water-loss stress.

Cuticular wax is the first barrier between plants and the environment. Here, the densities of cuticular wax crystals on the petals of eight rose cultivars were determined to be sparse; the crystals were mostly granular and only a few rod-shaped crystals were observed in 'Sweet'. The total contents and chemical compositions of waxes were significantly different among the rose varieties. The waxes were mainly composed of n-alkanes, iso-alananes and alkenes. Under water-loss stress, 'Diana' and 'Carola' cultivars, having high petal wax contents, had low water permeability levels, long vase lives, high relative water contents and low relative conductivity levels. However, the low wax contents of the 'Jubilance' and 'Candy Avalanche' cultivars resulted in high water permeability levels and short vase lives. Pearson correlation analyses showed the total wax content in petal epidermis was positively correlated with vase life. The data provide novel insights into the compositional variances in the cuticular waxes of rose petals and their impacts on cut rose vase lives.

Insect Cuticle Protein Nanoassemblies without Nonspecific Immune Response for Acute Methicillin-Resistant Staphylococcus aureus Pneumonia Remission.

The emergence and proliferation of methicillin-resistant Staphylococcus aureus (MRSA) pneumonia poses a significant global public health threat. Herein, the significant remission effect against acute MRSA pneumonia was realized through the insect cuticle protein (OfCPH-2) nanoassemblies without nonspecific immune response. The lung repair results could be attributed to the transforming of M1-type to M2-type macrophage polarization and the repression of Th17 cell differentiation in mice spleens through the intervention of OfCPH-2 nanoassemblies. These findings offer a valuable insight into the application of insect protein-based materials as effective antidrug resistant strain agents as well as a powerful strategy for acute MRSA pneumonia.

Identification and Functional Insights of Knickkopf Genes in the Larval Cuticle of Leptinotarsa decemlineata.

The Colorado potato beetle (Leptinotarsa decemlineata) is a major pest of potato crops. While Knickkopf (Knk) genes are essential for insect cuticle formation, their roles in pests like L. decemlineata remain unclear. This study aims to identify and characterize Knk genes in L. decemlineata and explore their functions in larval development and cuticle integrity. We used genomic and transcriptomic databases to identify LdKnk-family genes, validated through RT-PCR and RACE. Gene expression was analyzed at various developmental stages and tissues using qRT-PCR. RNA interference (RNAi) and Transmission electron microscopy (TEM) were applied to determine the functional roles of these genes. Four LdKnk-family genes were identified. Spatio-temporal expression analysis indicated significant gene expression during larval molting and pupal stages, especially in the epidermis. RNAi experiments showed that silencing LdKnk and LdKnk3-5' led to reduced larval weight, cuticle thinning, and increased mortality, while LdKnk3-FL knockdown caused abnormal cuticle thickening and molting disruptions. LdKnk2 knockdown increased epicuticle and endocuticle thickness without visible phenotypic changes. The study highlights the essential roles of LdKnk-family genes in maintaining cuticle structure and integrity, suggesting their potential as targets for RNAi-based pest control.

MaAzaR Influences Virulence of Metarhizium acridum against Locusta migratoria manilensis by Affecting Cuticle Penetration.

The entomopathogenic fungus (EPF) Metarhizium acridum is a typical filamentous fungus and has been used to control migratory locusts (Locusta migratoria manilensis). This study examines the impact of the Zn(II)2Cys6 transcription factor, MaAzaR, in the virulence of M. acridum. Disruption of MaAzaR (ΔMaAzaR) diminished the fungus's ability to penetrate the insect cuticle, thereby decreasing its virulence. The median lethal time (LT50) for the ΔMaAzaR strain increased by approximately 1.5 d compared to the wild-type (WT) strain when topically inoculated, simulating natural infection conditions. ΔMaAzaR compromises the formation, turgor pressure, and secretion of extracellular hydrolytic enzymes in appressoria. However, the growth ability of ΔMaAzaR within the hemolymph is not impaired; in fact, it grows better than the WT strain. Moreover, RNA-sequencing (RNA-Seq) analysis of ΔMaAzaR and WT strains grown for 20 h on locust hindwings revealed 87 upregulated and 37 downregulated differentially expressed genes (DEGs) in the mutant strain. Pathogen-host interaction database (PHI) analysis showed that about 40% of the total DEGs were associated with virulence, suggesting that MaAzaR is a crucial transcription factor that directly regulates the expression of downstream genes. This study identifies a new transcription factor involved in EPF cuticle penetration, providing theoretical support and genetic resources for the developing highly virulent strains.

A high-quality genome assembly reveals adaptations underlying glossy, wax-coated leaves in the heat-tolerant wild raspberry Rubus leucanthus.

With glossy, wax-coated leaves, Rubus leucanthus is one of the few heat-tolerant wild raspberry trees. To ascertain the underlying mechanism of heat tolerance, we generated a high-quality genome assembly with a genome size of 230.9 Mb and 24,918 protein-coding genes. Significantly expanded gene families were enriched in the flavonoid biosynthesis pathway and the circadian rhythm-plant pathway, enabling survival in subtropical areas by accumulating protective flavonoids and modifying photoperiodic responses. In contrast, plant-pathogen interaction and MAPK signaling involved in response to pathogens were significantly contracted. The well-known heat response elements (HSP70, HSP90, and HSFs) were reduced in R. leucanthus compared to two other heat-intolerant species, R. chingii and R. occidentalis, with transcriptome profiles further demonstrating their dispensable roles in heat stress response. At the same time, three significantly positively selected genes in the pathway of cuticular wax biosynthesis were identified, and may contribute to the glossy, wax-coated leaves of R. leucanthus. The thick, leathery, waxy leaves protect R. leucanthus against pathogens and herbivores, supported by the reduced R gene repertoire in R. leucanthus (355) compared to R. chingii (376) and R. occidentalis (449). Our study provides some insights into adaptive divergence between R. leucanthus and other raspberry species on heat tolerance.

Metabolic and gene-expression analyses reveal developmental dynamics of cutin deposition in pomegranate fruit grown under different environmental conditions.

The chemical and transcriptional changes in the cuticle of pomegranate (Punica granatum L.) fruit grown under different environmental conditions were studied. We collected fruit from three orchards located in different regions in Israel, each with a distinct microclimate. Fruit were collected at six phenological stages, and cutin monomers in the fruit cuticle were profiled by gas chromatography-mass spectrometry (GC-MS), along with qPCR transcript-expression analyses of selected cutin-related genes. While fruit phenotypes were comparable along development in all three orchards, principal component analyses of cutin monomer profiles suggested clear separation between cuticle samples of young green fruit to those of maturing fruit. Moreover, total cutin contents in green fruit were lower in the orchard characterized by a hot and dry climate compared to orchards with moderate temperatures. The variances detected in total cutin contents between orchards corresponded well with the expression patterns of BODYGUARD, a key biosynthetic gene operating in the cutin biosynthetic pathway. Based on our extraction protocols, we found that the cutin polyester that builds the pomegranate fruit cuticle accumulates some levels of gallic acid-the precursor of punicalagin, a well-known potent antioxidant metabolite in pomegranate fruit. The gallic acid was also one of the predominant metabolites contributing to the variability between developmental stages and orchards, and its accumulation levels were opposite to the expression patterns of the UGT73AL1 gene which glycosylates gallic acid to synthesize punicalagin. To the best of our knowledge, this is the first detailed composition of the cutin polyester that forms the pomegranate fruit cuticle.

A positive loop between relish and cuticle proteins and their roles in regulating AMPs expression during bacterial infection in Eriocheir sinensis.

Cuticle proteins (CPs) are the vital components of the cuticle and chitin lining covering the digestive tract of crustaceans. In this study, four new CP genes (designated as EsCP3, EsCP4, EsCP5, and EsCP8) were initially cloned and identified from the Chinese mitten crab Eriocheir sinensis. EsCP3/4/5/8 included 375, 411, 381, and 570 bp open reading frame encoding 124, 136, 126, and 189 amino acid proteins, respectively. Except for EsCP8, EsCP3/4/5 all contained a Chitin_bind_4 domain. EsCP3/4/5/8 were clustered into different groups in the phylogenetic tree. Quantitative real-time PCR results indicated that four EsCP genes have different patterns of tissue distribution. Changes in the expression levels of these four EsCP genes were observed in the intestine of crabs under Vibrio parahaemolyticus challenge. RNA interference assay showed that the knockdown of EsCPs in the intestine could inhibit the expression of antimicrobial peptides (AMPs), including crustins and anti-lipopolysaccharide factors. In addition, the knockdown of EsRelish in the intestine decreased the expression levels of these four EsCP genes. These results indicated that EsCPs were involved in regulating the expression of AMPs, and EsCPs were regulated by EsRelish.

Characterization of an RR-2 cuticle protein DcCP8 and its potential application based on SPc nanoparticle-wrapped dsRNA in Diaphorina citri.

BACKGROUND: The insect cuticle consists of chitin fibers and a protein matrix, which plays an important role in protecting the body from invasion of various pathogens and prevents water loss. Periodic synthesis and degradation of the cuticle is required for the growth and development of insects. Key genes involved in cuticle formation have long been considered a potential target for pest control. RESULTS: In this study, a member of the RR-2 subfamily of cuticular protein 8 (DcCP8) was identified from the Diaphorina citri genome database. Immunofluorescence analysis suggested that DcCP8 was mainly located in the Diaphorina citri exocuticle and can be induced to up-regulate 12 h following 20-hydroxyecdysone (20E) treatment. Silencing of DcCP8 by RNA interference (RNAi) significantly disrupted the metamorphosis to the adult stage, and improved the permeability of the cuticle. Transmission electron microscopy (TEM) analysis revealed that the synthesis of the exocuticle was impressed after silencing of DcCP8. Furthermore, the recombinant DcCP8 protein exhibited chitin-binding properties in vitro, down-regulation of DcCP8 significantly inhibited expression levels of chitin metabolism-related genes. Additionally, a sprayable RNAi method targeting DcCP8 based on star polycation (SPc) nanoparticles-wrapped double-stranded RNA (dsRNA) significantly increased Diaphorina citri mortality. Transcriptome sequencing further confirmed that genes associated with the endocytic pathway and immune response were up-regulated in Diaphorina citri after SPc treatment. CONCLUSIONS: The current study indicated that DcCP8 is critical for the formation of Diaphorina citri exocuticles, and lays a foundation for Diaphorina citri control based on large-scale dsRNA nanoparticles. © 2024 Society of Chemical Industry.

Effectiveness and biochemical impact of ozone gas and silica nanoparticles on Culex pipiens (Diptera: Culicidae).

Culex pipiens (Diptera: Culicidae) is a vector of many serious human diseases, and its control by the heavy use of chemical insecticides has led to the evolution of insecticide resistance and high environmental risks. Many safe alternatives, such as ozone gas (O3) and silica nanoparticles (silica NPs) can reduce these risks. Therefore, O3 and silica NPs were applied to 3rd larval instars of Cx. pipiens at different concentrations (100, 200, and 400 ppm) for different exposure times (1, 2, 3, and 5 min for O3 and 24, 48, and 72 h for silica NPs). The activity of some vital antioxidant enzymes as well as scanning electron microscopy of the body surface were also investigated. A positive correlation was observed between larval mortality % and the tested concentrations of O3 and silica NPs. O3 was more effective than silica NPs, it resulted in 92% mortality at 400 ppm for a short exposure time (5 min). O3-exposed larvae exhibited a significant increase in glutathione peroxidase, glutathione S-transferase, and catalase activities as well as the total antioxidant capacity. Scanning electron microscopy showing disruptive effects on the body surface morphology of ozone and silica NPs treated larvae. These results provide evidence that O3 and silica NPs have the potential for use as alternative vector control tools against Cx. pipiens.

How frequently are insects wounded in the wild? A case study using Drosophila melanogaster.

Wounding occurs across multicellular organisms. Wounds can affect host mobility and reproduction, with ecological consequences for competitive interactions and predator-prey dynamics. Wounds are also entry points for pathogens. An immune response is activated upon injury, resulting in the deposition of the brown-black pigment melanin in insects. Despite the abundance of immunity studies in the laboratory and the potential ecological and evolutionary implications of wounding, the prevalence of wounding in wild-collected insects is rarely systematically explored. We investigated the prevalence and potential causes of wounds in wild-collected Drosophilidae flies. We found that 31% of Drosophila melanogaster were wounded or damaged. The abdomen was the most frequently wounded body part, and females were more likely to have melanized patches on the ventral abdomen, compared with males. Encapsulated parasitoid egg frequency was approximately 10%, and just under 1% of Drosophilidae species had attached mites, which also caused wounds. Wounding is prevalent in D. melanogaster, likely exerting selection pressure on host immunity for two reasons: on a rapid and efficient wound repair and on responding efficiently to opportunistic infections. Wounding is thus expected to be an important driver of immune system evolution and to affect individual fitness and population dynamics.

Role of cuticular genes in the insect antimicrobial immune response.

Insects are established models for understanding host-pathogen interactions and innate immune mechanisms. The innate immune system in insects is highly efficient in recognizing and opposing pathogens that cause detrimental effects during infection. The cuticular layer which covers the superficial layer of the insect body participates in host defense and wound healing by inducing innate immune responses. Previous studies have started to address the involvement of cuticular genes in conferring resistance to insect pathogens, particularly those that infect by disrupting the insect cuticle. For example, the cuticular gene Transglutaminase (TG) in Drosophila melanogaster plays a structural role in cuticle formation and blood coagulation and also possesses immune properties against pathogenic infection. However, more information is becoming available about the immune function of other cuticular gene families in insects. In this review, we aim to highlight the recent advances in insect cuticular immunity and address the necessity of pursuing further research to fill the existing gaps in this important field of insect immunology. This information will lead to novel strategies for the efficient management of agricultural insect pests and vectors of plant and human disease.

Estimates of late Early Cretaceous atmospheric CO2 from Mongolia based on stomatal and isotopic analysis of Pseudotorellia.

PREMISE: The Aptian-Albian (121.4-100.5 Ma) was a greenhouse period with global temperatures estimated as 10-15°C warmer than pre-industrial conditions, so it is surprising that the most reliable CO2 estimates from this time are <1400 ppm. This low CO2 during a warm period implies a very high Earth-system sensitivity in the range of 6 to 9°C per CO2 doubling between the Aptian-Albian and today. METHODS: We applied a well-vetted paleo-CO2 proxy based on leaf gas-exchange principles (Franks model) to two Pseudotorellia species from three stratigraphically similar samples at the Tevshiin Govi lignite mine in central Mongolia (~119.7-100.5 Ma). RESULTS: Our median estimated CO2 concentration from the three respective samples was 2132, 2405, and 2770 ppm. The primary reason for the high estimated CO2 but with relatively large uncertainties is the very low stomatal density in both species, where small variations propagate to large changes in estimated CO2. Indeed, we found that at least 15 leaves are required before the aggregate estimated CO2 approaches that of the full data set. CONCLUSIONS: Our three CO2 estimates all exceeded 2000 ppm, translating to an Earth-system sensitivity (~3-5°C/CO2 doubling) that is more in keeping with the current understanding of the long-term climate system. Because of our large sample size, the directly measured inputs did not contribute much to the overall uncertainty in estimated CO2; instead, the inferred inputs were responsible for most of the overall uncertainty and thus should be scrutinized for their value choices.