Rapid response of nonstructural carbohydrate allocation and photosynthesis to short photoperiod, low temperature, or elevated CO2 in Pinus strobus.

During autumn, decreasing photoperiod and temperature temporarily perturb the balance between carbon uptake and carbon demand in overwintering plants, requiring coordinated adjustments in photosynthesis and carbon allocation to re-establish homeostasis. Here we examined adjustments of photosynthesis and allocation of nonstructural carbohydrates (NSCs) following a sudden shift to short photoperiod, low temperature, and/or elevated CO2 in Pinus strobus seedlings. Seedlings were initially acclimated to 14 h photoperiod (22/15°C day/night) and ambient CO2 (400 ppm) or elevated CO2 (800 ppm). Seedlings were then shifted to 8 h photoperiod for one of three treatments: no temperature change at ambient CO2 (22/15°C, 400 ppm), low temperature at ambient CO2 (12/5°C, 400 ppm), or no temperature change at elevated CO2 (22/15°C, 800 ppm). Short photoperiod caused all seedlings to exhibit partial nighttime depletion of starch. Short photoperiod alone did not affect photosynthesis. Short photoperiod combined with low temperature caused hexose accumulation and repression of photosynthesis within 24 h, followed by a transient increase in nonphotochemical quenching (NPQ). Under long photoperiod, plants grown under elevated CO2 exhibited significantly higher NSCs and photosynthesis compared to ambient CO2 plants, but carbon uptake exceeded sink capacity, leading to elevated NPQ; carbon sink capacity was restored and NPQ relaxed within 24 h after shift to short photoperiod. Our findings indicate that P. strobus rapidly adjusts NSC allocation, not photosynthesis, to accommodate short photoperiod. However, the combination of short photoperiod and low temperature, or long photoperiod and elevated CO2 disrupts the balance between photosynthesis and carbon sink capacity, resulting in increased NPQ to alleviate excess energy.

A long-read and short-read transcriptomics approach provides the first high-quality reference transcriptome and genome annotation for Pseudotsuga menziesii (Douglas-fir).

Douglas-fir (Pseudotsuga menziesii) is native to western North America. It grows in a wide range of environmental conditions and is an important timber tree. Although there are several studies on the gene expression responses of Douglas-fir to abiotic cues, the absence of high-quality transcriptome and genome data is a barrier to further investigation. Like for most conifers, the available transcriptome and genome reference dataset for Douglas-fir remains fragmented and requires refinement. We aimed to generate a highly accurate, and complete reference transcriptome and genome annotation. We deep-sequenced the transcriptome of Douglas-fir needles from seedlings that were grown under nonstress control conditions or a combination of heat and drought stress conditions using long-read (LR) and short-read (SR) sequencing platforms. We used 2 computational approaches, namely de novo and genome-guided LR transcriptome assembly. Using the LR de novo assembly, we identified 1.3X more high-quality transcripts, 1.85X more "complete" genes, and 2.7X more functionally annotated genes compared to the genome-guided assembly approach. We predicted 666 long noncoding RNAs and 12,778 unique protein-coding transcripts including 2,016 putative transcription factors. We leveraged the LR de novo assembled transcriptome with paired-end SR and a published single-end SR transcriptome to generate an improved genome annotation. This was conducted with BRAKER2 and refined based on functional annotation, repetitive content, and transcriptome alignment. This high-quality genome annotation has 51,419 unique gene models derived from 322,631 initial predictions. Overall, our informatics approach provides a new reference Douglas-fir transcriptome assembly and genome annotation with considerably improved completeness and functional annotation.

Drone-based physiological index reveals long-term acclimation and drought stress responses in trees.

Monitoring early tree physiological responses to drought is key to understanding progressive impacts of drought on forests and identifying resilient species. We combined drone-based multispectral remote sensing with measurements of tree physiology and environmental parameters over two growing seasons in a 100-y-old Pinus sylvestris forest subject to 17-y of precipitation manipulation. Our goal was to determine if drone-based photochemical reflectance index (PRI) captures tree drought stress responses and whether responses are affected by long-term acclimation. PRI detects changes in xanthophyll cycle pigment dynamics, which reflect increases in photoprotective non-photochemical quenching activity resulting from drought-induced photosynthesis downregulation. Here, PRI of never-irrigated trees was up to 10 times lower (higher stress) than PRI of irrigated trees. Long-term acclimation to experimental treatment, however, influenced the seasonal relationship between PRI and soil water availability. PRI also captured diurnal decreases in photochemical efficiency, driven by vapour pressure deficit. Interestingly, 5 years after irrigation was stopped for a subset of the irrigated trees, a positive legacy effect persisted, with lower stress responses (higher PRI) compared with never-irrigated trees. This study demonstrates the ability of remotely sensed PRI to scale tree physiological responses to an entire forest and the importance of long-term acclimation in determining current drought stress responses.

Chlorophyll a fluorescence illuminates a path connecting plant molecular biology to Earth-system science.

For decades, the dynamic nature of chlorophyll a fluorescence (ChlaF) has provided insight into the biophysics and ecophysiology of the light reactions of photosynthesis from the subcellular to leaf scales. Recent advances in remote sensing methods enable detection of ChlaF induced by sunlight across a range of larger scales, from using instruments mounted on towers above plant canopies to Earth-orbiting satellites. This signal is referred to as solar-induced fluorescence (SIF) and its application promises to overcome spatial constraints on studies of photosynthesis, opening new research directions and opportunities in ecology, ecophysiology, biogeochemistry, agriculture and forestry. However, to unleash the full potential of SIF, intensive cross-disciplinary work is required to harmonize these new advances with the rich history of biophysical and ecophysiological studies of ChlaF, fostering the development of next-generation plant physiological and Earth-system models. Here, we introduce the scale-dependent link between SIF and photosynthesis, with an emphasis on seven remaining scientific challenges, and present a roadmap to facilitate future collaborative research towards new applications of SIF.

Champions of winter survival: cold acclimation and molecular regulation of cold hardiness in evergreen conifers.

Evergreen conifers are champions of winter survival, based on their remarkable ability to acclimate to cold and develop cold hardiness. Counterintuitively, autumn cold acclimation is triggered not only by exposure to low temperature, but also by a combination of decreasing temperature, decreasing photoperiod and changes in light quality. These environmental cues control a network of signaling pathways that coordinate cold acclimation and cold hardiness in overwintering conifers, leading to cessation of growth, bud dormancy, freezing tolerance and changes in energy metabolism. Advances in genomic, transcriptomic and metabolomic tools for conifers have improved our understanding of how trees sense and respond to changes in temperature and light during cold acclimation and the development of cold hardiness, but there remain considerable gaps deserving further research in conifers. In the first section of this review, we focus on the physiological mechanisms used by evergreen conifers to adjust metabolism seasonally and to protect overwintering tissues against winter stresses. In the second section, we review how perception of low temperature and photoperiod regulate the induction of cold acclimation. Finally, we explore the evolutionary context of cold acclimation in conifers and evaluate challenges imposed on them by changing climate and discuss emerging areas of research in the field.

Decoding Gene Networks Modules That Explain the Recovery of Hymenoglossum cruentum Cav. After Extreme Desiccation.

Hymenoglossum cruentum (Hymenophyllaceae) is a poikilohydric, homoiochlorophyllous desiccation-tolerant (DT) epiphyte fern. It can undergo fast and frequent dehydration-rehydration cycles. This fern is highly abundant at high-humidity/low-light microenvironments within the canopy, although rapid changes in humidity and light intensity are frequent. The objective of this research is to identify genes associated to desiccation-rehydration cycle in the transcriptome of H. cruentum to better understand the genetic dynamics behind its desiccation tolerance mechanism. H. cruentum plants were subjected to a 7 days long desiccation-rehydration process and then used to identify key expressed genes associated to its capacity to dehydrate and rehydrate. The relative water content (RWC) and maximum quantum efficiency (F v/F m) of H. cruentum fronds decayed to 6% and 0.04, respectively, at the end of the desiccation stage. After re-watering, the fern showed a rapid recovery of RWC and F v/F m (ca. 73% and 0.8, respectively). Based on clustering and network analysis, our results reveal key genes, such as UBA/TS-N, DYNLL, and LHC, orchestrating intracellular motility and photosynthetic metabolism; strong balance between avoiding cell death and defense (CAT3, AP2/ERF) when dehydrated, and detoxifying pathways and stabilization of photosystems (GST, CAB2, and ELIP9) during rehydration. Here we provide novel insights into the genetic dynamics behind the desiccation tolerance mechanism of H. cruentum.

Variation in the phenology of photosynthesis among eastern white pine provenances in response to warming.

In higher-latitude trees, temperature and photoperiod control the beginning and end of the photosynthetically active season. Elevated temperature (ET) has advanced spring warming and delayed autumn cooling while photoperiod remains unchanged. We assessed the effects of warming on the length of the photosynthetically active season of three provenances of Pinus strobus L. seedlings from different latitudes, and evaluated the accuracy of the photochemical reflectance index (PRI) and the chlorophyll/carotenoid index (CCI) for tracking the predicted variation in spring and autumn phenology of photosynthesis among provenances. Seedlings from northern, local and southern P. strobus provenances were planted in a temperature-free-air-controlled enhancement (T-FACE) experiment and exposed to ET (+1.5/3°C; day/night). Over 18 months, we assessed photosynthetic phenology by measuring chlorophyll fluorescence, gas exchange, leaf spectral reflectance and pigment content. During autumn, all seedlings regardless of provenance followed the same sequence of phenological events with the initial downregulation of photosynthesis, followed by the modulation of non-photochemical quenching and associated adjustments of zeaxanthin pool sizes. However, the timing of autumn downregulation differed between provenances, with delayed onset in the southern provenance (SP) and earlier onset in the northern relative to the local provenance, indicating that photoperiod at the provenance origin is a dominant factor controlling autumn phenology. Experimental warming further delayed the downregulation of photosynthesis during autumn in the SP. A provenance effect during spring was also observed but was generally not significant. The vegetation indices PRI and CCI were both effective at tracking the seasonal variations of energy partitioning in needles and the differences of carotenoid pigments indicative of the stress status of needles. These results demonstrate that PRI and CCI can be useful tools for monitoring conifer phenology and for the remote monitoring of the length of the photosynthetically active season of conifers in a changing climate.

High-throughput drone-based remote sensing reliably tracks phenology in thousands of conifer seedlings.

Phenology is an important indicator of environmental variation and climate change impacts on tree responses. In conifers, monitoring phenology of photosynthesis through remote sensing has been unreliable, because needle foliage varies little throughout the year. This is challenging for modelling ecosystem carbon uptake and monitoring phenology for enhanced breeding (genomic selection) and forest health. Here, we demonstrate that drone-based carotenoid-sensitive spectral indices, such as the Chl/carotenoid index (CCI), can be used to track phenology in conifers by taking advantage of the close relationship between seasonally changing carotenoid levels and the variation of photosynthetic activity. Physiological ground measurements, including photosynthetic pigments and maximum quantum yield of Chl fluorescence, indicated that CCI tracked the variation of photosynthetic activity better than other vegetation indices for 30 white spruce seedlings measured over 1 yr. A machine-learning approach, using CCI derived from drone-based multispectral imagery, was used to model phenology of photosynthesis for the entire pedigree population (6000 seedlings). This high-throughput drone-based phenotyping approach is suitable for studying climate change impacts and environmental variation on the physiological status of thousands of field-grown conifers at unprecedented speed and scale.

Tracking the phenology of photosynthesis using carotenoid-sensitive and near-infrared reflectance vegetation indices in a temperate evergreen and mixed deciduous forest.

Photosynthetic phenology is an important indicator of annual gross primary productivity (GPP). Assessing photosynthetic phenology remotely is difficult for evergreen conifers as they remain green year-round. Carotenoid-based vegetation indices such as the photochemical reflectance index (PRI) and chlorophyll/carotenoid index (CCI) are promising tools to remotely track the invisible phenology of photosynthesis by assessing carotenoid pigment dynamics. PRI, CCI and the near-infrared reflectance of vegetation (NIRV ) index may act as proxies of photosynthetic efficiency (ɛ), an important parameter in light-use efficiency models, or direct proxies of photosynthesis. To understand the physiological mechanisms reflected by PRI and CCI and the ability of vegetation indices to act as proxies of photosynthetic activity for estimating GPP, we measured leaf pigment composition, PRI, CCI, NIRV and photosynthetic activity at the leaf and canopy scales over 2 years in an evergreen and mixed deciduous forest. PRI and CCI captured the large seasonal carotenoid/chlorophyll ratio changes and good relationships were observed between PRI-ɛ and CCI-photosynthesis and NIRV -photosynthesis. PRI-, CCI- and NIRV -based models effectively tracked observed seasonal GPP. We propose that carotenoid-based and near-infrared reflectance vegetation indices may provide useful proxies of photosynthetic activity and can improve remote sensing-based models of GPP in evergreen and deciduous forests.

A comparative gene co-expression analysis using self-organizing maps on two congener filmy ferns identifies specific desiccation tolerance mechanisms associated to their microhabitat preference.

BACKGROUND: Filmy-ferns (Hymenophyllaceae) are poikilohydric, homoiochlorophyllous desiccation-tolerant (DT) epiphytes. They can colonize lower and upper canopy environments of humid forest. Filmy-ferns desiccate rapidly (hours), contrasting with DT angiosperms (days/weeks). It has been proposed that desiccation tolerance in filmy-ferns would be associated mainly with constitutive features rather than induced responses during dehydration. However, we hypothesize that the inter-specific differences in vertical distribution would be associated with different dynamics of gene expression within the dehydration or rehydration phases. A comparative transcriptomic analysis with an artificial neural network was done on Hymenophyllum caudiculatum (restricted to lower canopy) and Hymenophyllum dentatum (reach upper canopy) during a desiccation/rehydration cycle. RESULTS: Raw reads were assembled into 69,599 transcripts for H. dentatum and 34,726 transcripts for H. caudiculatum. Few transcripts showed significant changes in differential expression (DE). H. caudiculatum had ca. twice DE genes than H. dentatum and higher proportion of increased-and-decreased abundance of genes occurs during dehydration. In contrast, the abundance of genes in H. dentatum decreased significantly when transitioning from dehydration to rehydration. According to the artificial neural network results, H. caudiculatum enhanced osmotic responses and phenylpropanoid related pathways, whilst H. dentatum enhanced its defense system responses and protection against high light stress. CONCLUSIONS: Our findings provide a deeper understanding of the mechanisms underlying the desiccation tolerance responses of two filmy ferns and the relationship between the species-specific response and the microhabitats these ferns occupy in nature.

Evidence for convergent sensing of multiple abiotic stresses in cyanobacteria.

BACKGROUND: Bacteria routinely utilize two-component signal transduction pathways to sense and alter gene expression in response to environmental cues. While cyanobacteria express numerous two-component systems, these pathways do not regulate all of the genes within many of the identified abiotic stress-induced regulons. METHODS: Electron transport inhibitors combined with western analysis and measurement of chlorophyll a fluorescent yield, using pulse amplitude modulation fluorometry, were used to detect the effect of a diverse range of abiotic stresses on the redox status of the photosynthetic electron transport chain and the accumulation and degradation of the Synechocystis sp. PCC 6803 DEAD box RNA helicase, CrhR. RESULTS: Alterations in CrhR abundance were tightly correlated with the redox poise of the electron transport chain between QA and cytochrome b6f, with reduction favoring CrhR accumulation. CONCLUSIONS: The results provide evidence for an alternative, convergent sensing mechanism mediated through the redox poise of QB/PQH2 that senses multiple, divergent forms of abiotic stress and regulates accumulation of CrhR. The RNA helicase activity of CrhR could then function as a post-translational effector to regulate downstream gene expression. GENERAL SIGNIFICANCE: The potential for a related system in Staphylococcus aureus and higher plant chloroplasts suggest convergent sensing mechanisms may be evolutionarily conserved and occur more widely than anticipated.

Differences in isoprenoid-mediated energy dissipation pathways between coastal and interior Douglas-fir seedlings in response to drought.

Plants have evolved energy dissipation pathways to reduce photooxidative damage under drought when photosynthesis is hampered. Non-volatile and volatile isoprenoids are involved in non-photochemical quenching of excess light energy and scavenging of reactive oxygen species. A better understanding of trees' ability to cope with and withstand drought stress will contribute to mitigate the negative effects of prolonged drought periods expected under future climate conditions. Therefore we investigated if Douglas-fir (Pseudotsuga menziesii(Mirb.)) provenances from habitats with contrasting water availability reveal intraspecific variation in isoprenoid-mediated energy dissipation pathways. In a controlled drought experiment with 1-year-old seedlings of an interior and a coastal Douglas-fir provenance, we assessed the photosynthetic capacity, pool sizes of non-volatile isoprenoids associated with the photosynthetic apparatus, as well as pool sizes and emission of volatile isoprenoids. We observed variation in the amount and composition of non-volatile and volatile isoprenoids among provenances, which could be linked to variation in photosynthetic capacity under drought. The coastal provenance exhibited an enhanced biosynthesis and emission of volatile isoprenoids, which is likely sustained by generally higher assimilation rates under drought. In contrast, the interior provenance showed an enhanced photoprotection of the photosynthetic apparatus by generally higher amounts of non-volatile isoprenoids and increased amounts of xanthophyll cycle pigments under drought. Our results demonstrate that there is intraspecific variation in isoprenoid-mediated energy dissipation pathways among Douglas-fir provenances, which may be important traits when selecting provenances suitable to grow under future climate conditions.

Foliar nitrogen metabolism of adult Douglas-fir trees is affected by soil water availability and varies little among provenances.

The coniferous forest tree Douglas-fir (Pseudotsuga menziesii) is native to the pacific North America, and is increasingly planted in temperate regions worldwide. Nitrogen (N) metabolism is of great importance for growth, resistance and resilience of trees. In the present study, foliar N metabolism of adult trees of three coastal and one interior provenance of Douglas-fir grown at two common gardens in southwestern Germany (Wiesloch, W; Schluchsee, S) were characterized in two subsequent years. Both the native North American habitats of the seed sources and the common garden sites in Germany differ in climate conditions. Total and mineral soil N as well as soil water content were higher in S compared to W. We hypothesized that i) provenances differ constitutively in N pool sizes and composition, ii) N pools are affected by environmental conditions, and iii) that effects of environmental factors on N pools differ among interior and coastal provenances. Soil water content strongly affected the concentrations of total N, soluble protein, total amino acids (TAA), arginine and glutamate. Foliar concentrations of total N, soluble protein, structural N and TAA of trees grown at W were much higher than in trees at S. Provenance effects were small but significant for total N and soluble protein content (interior provenance showed lowest concentrations), as well as arginine, asparagine and glutamate. Our data suggest that needle N status of adult Douglas-fir is independent from soil N availability and that low soil water availability induces a re-allocation of N from structural N to metabolic N pools. Small provenance effects on N pools suggest that local adaptation of Douglas-fir is not dominated by N conditions at the native habitats.

Drought effects on root and needle terpenoid content of a coastal and an interior Douglas fir provenance.

Douglas fir (Pseudotsuga menziesii) is a conifer species that stores large amounts of terpenoids, mainly monoterpenoids in resin ducts of various tissues. The effects of drought on stored leaf terpenoid concentrations in trees are scarcely studied and published data are partially controversial, since reduced, unaffected or elevated terpenoid contents due to drought have been reported. Even less is known on the effect of drought on root terpenoids. In the present work, we investigated the effect of reduced water availability on the terpenoid content in roots and needles of Douglas fir seedlings. Two contrasting Douglas fir provenances were studied: an interior provenance (var. glauca) with assumed higher drought resistance, and a coastal provenance (var. menziesii) with assumed lower drought resistance. We tested the hypothesis that both provenances show specific patterns of stored terpenoids and that the patterns will change in response to drought in both, needles and roots. We further expected stronger changes in the less drought tolerant coastal provenance. For this purpose, we performed an experiment under controlled conditions, in which the trees were exposed to moderate and severe drought stress. According to our expectations, the study revealed clear provenance-specific terpenoid patterns in needles. However, such patterns were not detected in the roots. Drought slightly increased the needle terpenoid contents of the coastal but not of the interior provenance. We also observed increased terpenoid abundance mainly in roots of the moderately stressed coastal provenance. Overall, from the observed provenance-specific reactions with increased terpenoid levels in trees of the coastal origin in response to drought, we conclude on functions of terpenoids for abiotic stress tolerance that might be fulfilled by other, constitutively expressed mechanisms in drought-adapted interior provenances.

Variation in short-term and long-term responses of photosynthesis and isoprenoid-mediated photoprotection to soil water availability in four Douglas-fir provenances.

For long-lived forest tree species, the understanding of intraspecific variation among populations and their response to water availability can reveal their ability to cope with and adapt to climate change. Dissipation of excess excitation energy, mediated by photoprotective isoprenoids, is an important defense mechanism against drought and high light when photosynthesis is hampered. We used 50-year-old Douglas-fir trees of four provenances at two common garden experiments to characterize provenance-specific variation in photosynthesis and photoprotective mechanisms mediated by essential and non-essential isoprenoids in response to soil water availability and solar radiation. All provenances revealed uniform photoprotective responses to high solar radiation, including increased de-epoxidation of photoprotective xanthophyll cycle pigments and enhanced emission of volatile monoterpenes. In contrast, we observed differences between provenances in response to drought, where provenances sustaining higher CO2 assimilation rates also revealed increased water-use efficiency, carotenoid-chlorophyll ratios, pools of xanthophyll cycle pigments, ß-carotene and stored monoterpenes. Our results demonstrate that local adaptation to contrasting habitats affected chlorophyll-carotenoid ratios, pool sizes of photoprotective xanthophylls, ß-carotene, and stored volatile isoprenoids. We conclude that intraspecific variation in isoprenoid-mediated photoprotective mechanisms contributes to the adaptive potential of Douglas-fir provenances to climate change.

A remotely sensed pigment index reveals photosynthetic phenology in evergreen conifers.

In evergreen conifers, where the foliage amount changes little with season, accurate detection of the underlying "photosynthetic phenology" from satellite remote sensing has been difficult, presenting challenges for global models of ecosystem carbon uptake. Here, we report a close correspondence between seasonally changing foliar pigment levels, expressed as chlorophyll/carotenoid ratios, and evergreen photosynthetic activity, leading to a "chlorophyll/carotenoid index" (CCI) that tracks evergreen photosynthesis at multiple spatial scales. When calculated from NASA's Moderate Resolution Imaging Spectroradiometer satellite sensor, the CCI closely follows the seasonal patterns of daily gross primary productivity of evergreen conifer stands measured by eddy covariance. This discovery provides a way of monitoring evergreen photosynthetic activity from optical remote sensing, and indicates an important regulatory role for carotenoid pigments in evergreen photosynthesis. Improved methods of monitoring photosynthesis from space can improve our understanding of the global carbon budget in a warming world of changing vegetation phenology.

Fast detection of leaf pigments and isoprenoids for ecophysiological studies, plant phenotyping and validating remote-sensing of vegetation.

Rapid developments in remote-sensing of vegetation and high-throughput precision plant phenotyping promise a range of real-life applications using leaf optical properties for non-destructive assessment of plant performance. Use of leaf optical properties for assessing plant performance requires the ability to use photosynthetic pigments as proxies for physiological properties and the ability to detect these pigments fast, reliably and at low cost. We describe a simple and cost-effective protocol for the rapid analysis of chlorophylls, carotenoids and tocopherols using high-performance liquid chromatography (HPLC). Many existing methods are based on the expensive solvent acetonitrile, take a long time or do not include lutein epoxide and α-carotene. We aimed to develop an HPLC method which separates all major chlorophylls and carotenoids as well as lutein epoxide, α-carotene and α-tocopherol. Using a C30 -column and a mobile phase with a gradient of methanol, methyl-tert-butyl-ether (MTBE) and water, our method separates the above pigments and isoprenoids within 28 min. The broad applicability of our method is demonstrated using samples from various plant species and tissue types, e.g. leaves of Arabidopsis and avocado plants, several deciduous and conifer tree species, various crops, stems of parasitic dodder, fruit of tomato, roots of carrots and Chlorella algae. In comparison to previous methods, our method is very affordable, fast and versatile and can be used to analyze all major photosynthetic pigments that contribute to changes in leaf optical properties and which are of interest in most ecophysiological studies.

Elevated Temperature and CO2 Stimulate Late-Season Photosynthesis But Impair Cold Hardening in Pine.

Rising global temperature and CO2 levels may sustain late-season net photosynthesis of evergreen conifers but could also impair the development of cold hardiness. Our study investigated how elevated temperature, and the combination of elevated temperature with elevated CO2, affected photosynthetic rates, leaf carbohydrates, freezing tolerance, and proteins involved in photosynthesis and cold hardening in Eastern white pine (Pinus strobus). We designed an experiment where control seedlings were acclimated to long photoperiod (day/night 14/10 h), warm temperature (22°C/15°C), and either ambient (400 µL L-1) or elevated (800 µmol mol-1) CO2, and then shifted seedlings to growth conditions with short photoperiod (8/16 h) and low temperature/ambient CO2 (LTAC), elevated temperature/ambient CO2 (ETAC), or elevated temperature/elevated CO2 (ETEC). Exposure to LTAC induced down-regulation of photosynthesis, development of sustained nonphotochemical quenching, accumulation of soluble carbohydrates, expression of a 16-kD dehydrin absent under long photoperiod, and increased freezing tolerance. In ETAC seedlings, photosynthesis was not down-regulated, while accumulation of soluble carbohydrates, dehydrin expression, and freezing tolerance were impaired. ETEC seedlings revealed increased photosynthesis and improved water use efficiency but impaired dehydrin expression and freezing tolerance similar to ETAC seedlings. Sixteen-kilodalton dehydrin expression strongly correlated with increases in freezing tolerance, suggesting its involvement in the development of cold hardiness in P. strobus Our findings suggest that exposure to elevated temperature and CO2 during autumn can delay down-regulation of photosynthesis and stimulate late-season net photosynthesis in P. strobus seedlings. However, this comes at the cost of impaired freezing tolerance. Elevated temperature and CO2 also impaired freezing tolerance. However, unless the frequency and timing of extreme low-temperature events changes, this is unlikely to increase risk of freezing damage in P. strobus seedlings.