Non-linear response of Norway spruce to climate variation along elevational and age gradients in the Carpathians.

Climate change, namely increased warming coupled with a rise in extreme events (e.g., droughts, storms, heatwaves), is negatively affecting forest ecosystems worldwide. In these ecosystems, growth dynamics and biomass accumulation are driven mainly by environmental constraints, inter-tree competition, and disturbance regimes. Usually, climate-growth relationships are assessed by linear correlation due to the simplicity and straightforwardness of modeling. However, applying this method may bias results, since the ecological and physiological responses of trees to environmental factors are non-linear, and usually bell-shaped. In the Eastern Carpathian, Norway spruce is at the southeasternmost edge of its natural occurrence; this region is thus potentially vulnerable to climate change. A non-linear assessment of climate-growth relationships using machine-learning techniques for Norway spruce in this area had not been conducted prior to this study. To address this knowledge gap, we analyzed a large tree-ring network from 158 stands, with over 3000 trees of varying age distributed along an elevational gradient. Our results showed that non-linearity in the growth-climate response of spruce was season-specific: temperatures from the previous autumn and current growing season, along with water availability during winter, induced a bell-shaped response. Moreover, we found that at low elevations, spruce growth was mainly limited by water availability in the growing season, while winter temperatures are likely to have had a slight influence along the entire elevational gradient. Furthermore, at elevations lower than 1400 m, spruce trees were also found to be sensitive to previous autumn water availability. Overall, our results shed new light on the response of Norway spruce to climate in the Carpathians, which may aid in management decisions.

Identifying drivers of non-stationary climate-growth relationships of European beech.

The future performance of the widely abundant European beech (Fagus sylvatica L.) across its ecological amplitude is uncertain. Although beech is considered drought-sensitive and thus negatively affected by drought events, scientific evidence indicating increasing drought vulnerability under climate change on a cross-regional scale remains elusive. While evaluating changes in climate sensitivity of secondary growth offers a promising avenue, studies from productive, closed-canopy forests suffer from knowledge gaps, especially regarding the natural variability of climate sensitivity and how it relates to radial growth as an indicator of tree vitality. Since beech is sensitive to drought, we in this study use a drought index as a climate variable to account for the combined effects of temperature and water availability and explore how the drought sensitivity of secondary growth varies temporally in dependence on growth variability, growth trends, and climatic water availability across the species' ecological amplitude. Our results show that drought sensitivity is highly variable and non-stationary, though consistently higher at dry sites compared to moist sites. Increasing drought sensitivity can largely be explained by increasing climatic aridity, especially as it is exacerbated by climate change and trees' rank progression within forest communities, as (co-)dominant trees are more sensitive to extra-canopy climatic conditions than trees embedded in understories. However, during the driest periods of the 20th century, growth showed clear signs of being decoupled from climate. This may indicate fundamental changes in system behavior and be early-warning signals of decreasing drought tolerance. The multiple significant interaction terms in our model elucidate the complexity of European beech's drought sensitivity, which needs to be taken into consideration when assessing this species' response to climate change.

Early warning signals indicate climate change-induced stress in Norway spruce in the Eastern Carpathians.

Climate change is affecting forest ecosystems globally, in particular through warming as well as increases in the frequency and intensity of extreme events. Norway spruce (Picea abies (L.) Karst.) is one of the most important coniferous tree species in Europe. In recent extremely dry years in Central Europe, spruce suffered and large dieback has been observed. In parts of Eastern Europe, however, no large-scale decline in spruce has been reported so far, though anticipated changes in climate pose the question how the future of these forests may look like. To assess the current state of spruce forests in Eastern Europe, we established a tree-ring network consisting of 157 Norway spruce chronologies (from >3000 trees) of different ages distributed along elevational transects in the Eastern Carpathians, Romania. We evaluated early warning signals of climate change-induced stress, i.e. (1) growth decline, (2) increased sensitivity of tree growth (assessed over the statistics first-order autocorrelation and standard deviation), and (3) increased growth synchrony. A pronounced growth decline was observed over the last two decades, which was strongest in younger stands and at lower elevations. However, growth sensitivity and synchrony did not show consistent patterns, suggesting that forest decline may not be immediately imminent. Overall, our findings highlight an increased vulnerability of spruce in the Eastern Carpathians. With ongoing climate change, spruce dieback may be expected in this part of Europe as well.

Incorporating high-resolution climate, remote sensing and topographic data to map annual forest growth in central and eastern Europe.

To enhance our understanding of forest carbon sequestration, climate change mitigation and drought impact on forest ecosystems, the availability of high-resolution annual forest growth maps based on tree-ring width (TRW) would provide a significant advancement to the field. Site-specific characteristics, which can be approximated by high-resolution Earth observation by satellites (EOS), emerge as crucial drivers of forest growth, influencing how climate translates into tree growth. EOS provides information on surface reflectance related to forest characteristics and thus can potentially improve the accuracy of forest growth models based on TRW. Through the modelling of TRW using EOS, climate and topography data, we showed that species-specific models can explain up to 52 % of model variance (Quercus petraea), while combining different species results in relatively poor model performance (R2 = 13 %). The integration of EOS into models based solely on climate and elevation data improved the explained variance by 6 % on average. Leveraging these insights, we successfully generated a map of annual TRW for the year 2021. We employed the area of applicability (AOA) approach to delineate the range in which our models are deemed valid. The calculated AOA for the established forest-type models was 73 % of the study region, indicating robust spatial applicability. Notably, unreliable predictions predominantly occurred in the climate margins of our dataset. In conclusion, our large-scale assessment underscores the efficacy of combining climate, EOS and topographic data to develop robust models for mapping annual TRW. This research not only fills a critical void in the current understanding of forest growth dynamics but also highlights the potential of integrated data sources for comprehensive ecosystem assessments.

Surface ozone risk to human health and vegetation in tropical region: The case of Thailand.

Tropospheric ozone (O3) is a threat to vegetation and human health over the world, in particular in Asia. Knowledge on O3 impacts on tropical ecosystems is still very limited. An O3 risk assessment to crops, forests, and people from 25 monitoring stations across the tropical and subtropical Thailand during 2005-2018 showed that 44% of sites exceeded the critical levels (CLs) of SOMO35 (i.e., the annual Sum Of daily maximum 8-h Means Over 35 ppb) for human health protection. The concentration-based AOT40 CL (i.e., sum of the hourly exceedances above 40 ppb for daylight hours during the assumed growing season) was exceeded at 52% and 48% of the sites where the main crops rice and maize are present, respectively, and at 88% and 12% of the sites where evergreen or deciduous forests are present, respectively. The flux-based metric PODY (i.e., Phytotoxic Ozone Dose above a threshold Y of uptake) was calculated and was found to exceed the CLs at 1.0%, 1.5%, 20.0%, 1.5%, 0% and 68.0% of the sites where early rice, late rice, early maize, late maize, evergreen forests, and deciduous forests can grow, respectively. Trend analysis indicated that AOT40 increased over the study period (+5.9% year-1), while POD1 decreased (- 5.3% year-1), suggesting that the role of climate change in affecting the environmental factors that control stomatal uptake cannot be neglected. These results contribute novel knowledge on O3 threat to human health, forest productivity, and food security in tropical and subtropical areas.

The Response of Beech (Fagus sylvatica L.) Populations to Climate in the Easternmost Sites of Its European Distribution.

In the context of forecasted climate change scenarios, the growth of forest tree species at their distribution margin is crucial to adapt current forest management strategies. Analyses of beech (Fagus sylvatica L.) growth have shown high plasticity, but easternmost beech populations have been rarely studied. To describe the response of the marginal beech population to the climate in the far east sites of its distribution, we first compiled new tree ring width chronologies. Then we analyzed climate-growth relationships for three marginal beech populations in the Republic of Moldova. We observed a relatively high growth rate in the marginal populations compared to core distribution sites. Our analyses further revealed a distinct and significant response of beech growth to all climatic variables, assessing for the first time the relationship between growth and vapor pressure deficit (VPD) which described how plant growth responds to drought. These results highlight that accumulated water deficit is an essential limiting factor of beech growth in this region. In conclusion, beech growth in the easternmost marginal population is drought-limited, and the sensitivity to VPD will need to be considered in future studies to update the forest management of other economic and ecologically important species.

Do Different Tree-Ring Proxies Contain Different Temperature Signals? A Case Study of Norway Spruce (Picea abies (L.) Karst) in the Eastern Carpathians.

One of the most important proxy archives for past climate variation is tree rings. Tree-ring parameters offer valuable knowledge regarding how trees respond and adapt to environmental changes. Trees encode all environmental changes in different tree-ring parameters. In this study, we analyzed how air temperature is encoded in different Norway spruce tree-ring proxies along an altitude gradient in an intramountain valley of the Carpathians. The study area, in the Gheorgheni region, Romania (Eastern Carpathians), has a mountain climate with a frequent temperature inversion in winter. The climate−growth relationship was analyzed for two contrasting altitudes: low elevation, i.e., below 1000 m a.s.l., and high elevation, i.e., above 1500 m a.s.l. Two local weather stations, one in the valley and the other on the upper part of the mountains, provide daily temperatures (Joseni­750 m a.s.l. and Bucin­1282 m a.s.l.). The bootstrap Pearson correlation between cumulative daily temperature data and three tree-ring proxies (tree-ring width­TRW, basal area increment­BAI, and blue intensity­BI) was computed for each series. The results show that elevation modulates the climate response pattern in the case of BI, and remains relatively similar for TRW and BAI. The winter temperature's positive influence on spruce growth was observed in both TRW and BAI chronologies. Additionally, the BAI chronology highlights a positive relationship with summer temperature. The highest correlation coefficient (r = 0.551, p < 0.05, n = 41) was recorded between BI residual chronology from high elevation series and summer/autumn temperature from the upper-part weather station for a cumulative period of 59 days (the second half of August to the beginning of October). Our results show that, for this intramountain valley of the Eastern Carpathians, different tree-ring proxies capture different climate signals.

Geographical Distribution of Three Forest Invasive Beetle Species in Romania.

Ips duplicatus (Sahlberg, 1836), Xylosandrus germanus (Blandford, 1894) and Neoclytus acuminatus (Fabricius, 1775) are invasive species reported in Romania, but their current distribution is poorly known. The research aim was to provide new information on this issue. A survey was conducted over the period 2015-2017 in 82 locations, using flight-interception traps and bottle traps, baited with different attractants. Data obtained in our other unpublished studies were also taken into account. A total of 35,136 I. duplicatus beetles were collected in 30 survey locations. The highest captures were in the log yards of some factories processing logs of Norway spruce (Picea abies (L.) H. Karst.). Considering all known records so far, most of these are in the eastern part of Romania, where an outbreak took place during the years 2005-2014, mainly in spruce stands growing outside their natural range. During the survey, 4259 specimens of X. germanus were collected in 35 locations, but in our other studies the species was found in 13 additional places. It was collected at altitudes of 18-1200 m, and the largest catches were from beech stands, growing at 450-950 m. N. acuminatus was found in only six locations, in the western and southern parts of the country, at low altitudes, in tree stands composed of Fraxinus excelsior L., Quercus spp. and other broadleaf species, as well as in broadleaf log yards. The results suggest that I. duplicatus is established in most parts of the Norway spruce's range, X. germanus is still spreading in the country, with some areas having quite high populations, while N. acuminatus is present only in the warmest regions of the country.

What Is the Force-per-Molecule Inside a Biomaterial Having Randomly Oriented Units?

Both synthetic and natural protein-based materials are made of randomly oriented cross-linked molecules. Here we introduce a coarse-grained approach to estimate the average force-per-molecule for materials made from globular proteins. Our approach has three steps: placement of molecules inside a unit volume, cross-linking, and trimming to remove the protein domains that do not participate to the force response. Following this procedure, we estimate the number of active domains per cross-section area, that allows for a direct calculation of the force-per-domain. Among the variables considered, we found that concentration was the most sensitive parameter. We then synthesized protein hydrogels made from BSA and polyprotein L and measured the stresses that these materials can withstand. We found that forces-per-molecules of up to 17 pN per domain can be obtained experimentally using protein hydrogels. Our approach represents an important step toward understanding the scaling of tension in biomaterials.

Mechanical regulation of talin through binding and history-dependent unfolding.

Talin is a force-sensing multidomain protein and a major player in cellular mechanotransduction. Here, we use single-molecule magnetic tweezers to investigate the mechanical response of the R8 rod domain of talin. We find that under various force cycles, the R8 domain of talin can display a memory-dependent behavior: At the same low force (<10 pN), the same protein molecule shows vastly different unfolding kinetics. This history-dependent behavior indicates the evolution of a unique force-induced native state. We measure through mechanical unfolding that talin R8 domain binds one of its ligands, DLC1, with much higher affinity than previously reported. This strong interaction can explain the antitumor response of DLC1 by regulating inside-out activation of integrins. Together, our results paint a complex picture for the mechanical unfolding of talin in the physiological range and a new mechanism of function of DLC1 to regulate inside-out activation of integrins.

Nonexponential kinetics captured in sequential unfolding of polyproteins over a range of loads.

While performing under mechanical loads in vivo, polyproteins are vitally involved in cellular mechanisms such as regulation of tissue elasticity and mechano-transduction by unfolding their comprising domains and extending them. It is widely thought that the process of sequential unfolding of polyproteins follows an exponential kinetics as the individual unfolding events exhibit identical and identically distributed (iid) Poisson behavior. However, it was shown that under high loads, the sequential unfolding kinetics displays nonexponential kinetics that alludes to aging by a subdiffusion process. Statistical order analysis of this kinetics indicated that the individual unfolding events are not iid, and cannot be defined as a Poisson (memoryless) process. Based on numerical simulations it was argued that this behavior becomes less pronounced with lowering the load, therefore it is to be expected that polyproteins unfolding under lower forces will follow a Poisson behavior. This expectation serves as the motivation of the current study, in which we investigate the effect of force lowering on the unfolding kinetics of Poly-L8 under varying loads, specifically high (150, 100 â€‹pN) and moderate-low (45, 30, 20 â€‹pN) forces. We found that a hierarchy among the unfolding events still exists even under low loads, again resulting in nonexponential behavior. We observe that analyzing the dwell-time distributions with stretched-exponentials and power laws give rise to different phenomenological trends. Using statistical order analysis, we demonstrated that even under the lowest load, the sequential unfolding cannot be considered as iid, in accord with the power law distribution. Additional free energy analysis revealed the contribution of the unfolded segments elasticity that scales with the force on the overall one-dimensional contour of the energy landscape, but more importantly, it discloses the hierarchy within the activation barriers during sequential unfolding that account for the observed nonexponentiality.

Jet stream position explains regional anomalies in European beech forest productivity and tree growth.

The mechanistic pathways connecting ocean-atmosphere variability and terrestrial productivity are well-established theoretically, but remain challenging to quantify empirically. Such quantification will greatly improve the assessment and prediction of changes in terrestrial carbon sequestration in response to dynamically induced climatic extremes. The jet stream latitude (JSL) over the North Atlantic-European domain provides a synthetic and robust physical framework that integrates climate variability not accounted for by atmospheric circulation patterns alone. Surface climate impacts of north-south summer JSL displacements are not uniform across Europe, but rather create a northwestern-southeastern dipole in forest productivity and radial-growth anomalies. Summer JSL variability over the eastern North Atlantic-European domain (5-40E) exerts the strongest impact on European beech, inducing anomalies of up to 30% in modelled gross primary productivity and 50% in radial tree growth. The net effects of JSL movements on terrestrial carbon fluxes depend on forest density, carbon stocks, and productivity imbalances across biogeographic regions.

Kinetic Method of Producing Pores Inside Protein-Based Biomaterials without Compromising Their Structural Integrity.

Hydrogels made from globular proteins cross-linked covalently into a stable network are becoming an important type of biomaterial, with applications in artificial tissue design and cell culture scaffolds, and represent a promising system to study the mechanical and biochemical unfolding of proteins in crowded environments. Due to the small size of the globular protein domains, typically 2-5 nm, the primary network allows for a limited transfer of protein molecules and prevents the passing of particles and aggregates with dimensions over 100 nm. Here, we demonstrate a method to produce protein materials with micrometer-sized pores and increased permeability. Our approach relies on forming two competing networks: a covalent network made from cross-linked bovine serum albumin (BSA) proteins via a light-activated reaction and a physical network triggered by the aggregation of a polysaccharide, alginate, in the presence of Ca2+ ions. By fine-tuning the reaction times, we produce porous-protein hydrogels that retain the mechanical characteristics of their less-porous counterparts. We further describe a simple model to investigate the kinetic balance between the nucleation of alginate and cross-linking of BSA molecules and find the upper rate of the alginate aggregation reaction driving pore formation. By enabling a more significant permeability for protein-based materials without compromising their mechanical response, our method opens new vistas into studying protein-protein interactions and cell growth and designing novel affinity methods.

Comparative photophysical properties of some widely used fluorescent proteins under two-photon excitation conditions.

Understanding the photophysical properties of fluorescent proteins (FPs), such as emission and absorption spectra, molecular brightness, photostability, and photo-switching, is critical to the development of criteria for their selection as tags for fluorescent-based biological applications. While two-photon excitation imaging techniques have steadily gained popularity - due to comparatively deeper penetration depth, reduced out-of-focus photobleaching, and wide separation between emission spectra and two-photon excitation spectra -, most studies reporting on the photophysical properties of FPs tend to remain focused on single-photon excitation. Here, we report our investigation of the photophysical properties of several commonly used fluorescent proteins using two-photon microscopy with spectral resolution in both excitation and emission. Our measurements indicate that not only the excitation (and sometimes emission) spectra of FPs may be markedly different between single-photon and two-photon excitation, but also their relative brightness and their photo-stability. A good understanding of the photophysical properties of FPs under two-photon excitation is essential for choosing the right tag(s) for a desired experiment.

Impact of ground-level ozone on Mediterranean forest ecosystems health.

Given the high ozone concentrations observed in the Mediterranean region during summer, it is crucial to extend our knowledge on the potential ozone impacts on forest health with in situ studies, especially to protect typical endemic forests of the Mediterranean basin. This study is focused on ozone measurements and exposures over the Eastern Adriatic coast and on the calculation of different O3 metrics, i.e., accumulated exposure AOT40 (AOT40dir, AOT40ICP, AOT40pheno) and stomatal O3 fluxes with an hourly threshold of uptake (Y) to represent the detoxification capacity of trees (PODY, with Y = 0, 1, 2 nmol O3 m-2 s-1) used for forest protection. Finally, we provide an assessment of the relationships between the forest response indicators and environmental variables. Passive ozone measurements and monitoring of forest health indicators, namely growth and crown defoliation, were performed for Quercus ilex, Quercus pubescens, Pinus halepensis, and Pinus nigra forests. Results showed that, for all the analysed species, ozone levels were close to reached the upper plausibility limits for passive monitoring of air quality at forest sites (100 ppb), with the highest values found on P. halepensis in the summer period. O3 metrics based on exposure were found to be higher in pine plots than in oak plots, while the highest values of uptake-based metrics were found on P. nigra. Regarding relationships between environmental variables and forest-health response indicators, the crown defoliation was significantly correlated with the soil water content at various depth while the tree growth was correlated with the different O3 metrics. The most important predictors affecting tree growth of Q. pubescens and Q. ilex were AOT40pheno and AOT40dir and POD0 for P. nigra.

Climate warming induced synchronous growth decline in Norway spruce populations across biogeographical gradients since 2000.

Covering large parts of Europe, Norway spruce (Picea abies L Karst.) plays an important role in the adaptation strategy of forest services to future climate change. Although dendroecology can provide valuable information on the past relationships between tree growth and climate, most previous studies were biased towards species-specific distribution limits, where old individuals grow slowly under extreme conditions. In the present study, we investigated the growth variability and climate sensitivity of 2851 Norway spruce trees along longitudinal (E 12-26°), latitudinal (N 45-51°), and elevation (118-1591 m a.s.l.) gradients in central-eastern Europe. We reveal that summer weather significantly affects the radial growth of spruce trees, but the effects strongly vary along biogeographical gradients. Extreme summer heatwaves in 2000 and 2003 reduced the growth rates by 10-35%, most pronounced in the southern Carpathians. In contrast to the population in the Czech Republic, climate warming induced a synchronous decline in the growth rates across biogeographical gradients in the Carpathian arc. By demonstrating the increased vulnerability of Norway spruce under warmer climate conditions, we recommended that the forest services and conservation managers replace or admix monocultures of this species with more drought-resilient mixtures including fir, beech and other broadleaved species.

Does protein unfolding play a functional role in vivo?

Unfolding and refolding of multidomain proteins under force have yet to be recognized as a major mechanism of function for proteins in vivo. In this review, we discuss the inherent properties of multidomain proteins under a force vector from a structural and functional perspective. We then characterize three main systems where multidomain proteins could play major roles through mechanical unfolding: muscular contraction, cellular mechanotransduction, and bacterial adhesion. We analyze how key multidomain proteins for each system can produce a gain-of-function from the perspective of a fine-tuned quantized response, a molecular battery, delivery of mechanical work through refolding, elasticity tuning, protection and exposure of cryptic sites, and binding-induced mechanical changes. Understanding how mechanical unfolding and refolding affect function will have important implications in designing mechano-active drugs against conditions such as muscular dystrophy, cancer, or novel antibiotics.

Cation-induced shape programming and morphing in protein-based hydrogels.

Smart materials that are capable of memorizing a temporary shape, and morph in response to a stimulus, have the potential to revolutionize medicine and robotics. Here, we introduce an innovative method to program protein hydrogels and to induce shape changes in aqueous solutions at room temperature. We demonstrate our approach using hydrogels made from serum albumin, the most abundant protein in the blood plasma, which are synthesized in a cylindrical or flower shape. These gels are then programmed into a spring or a ring shape, respectively. The programming is performed through a marked change in stiffness (of up to 17-fold), induced by adsorption of Zn2+ or Cu2+ cations. We show that these programmed biomaterials can then morph back into their original shape, as the cations diffuse outside the hydrogel material. The approach demonstrated here represents an innovative strategy to program protein-based hydrogels to behave as actuators.

Binding-Induced Stabilization Measured on the Same Molecular Protein Substrate Using Single-Molecule Magnetic Tweezers and Heterocovalent Attachments.

Binding-induced mechanical stabilization plays key roles in proteins involved in muscle contraction, cellular mechanotransduction, or bacterial adhesion. Because of the vector nature of force, single-molecule force spectroscopy techniques are ideal for measuring the mechanical unfolding of proteins. However, current approaches are still prone to calibration errors between experiments and geometrical variations between individual tethers. Here, we introduce a single-molecule assay based on magnetic tweezers and heterocovalent attachment, which can measure the binding of the substrate-ligand using the same protein molecule. We demonstrate this approach with protein L, a model bacterial protein which has two binding interfaces for the same region of kappa-light chain antibody ligands. Engineered molecules with eight identical domains of protein L between a HaloTag and a SpyTag were exposed to repeated unfolding-refolding cycles at forces up to 100 pN for several hours at a time. The unfolding behavior of the same protein was measured in solution buffers with different concentrations of antibody ligands. With increasing antibody concentration, an increasing number of protein L domains became more stable, indicative of ligand binding and mechanical reinforcement. Interestingly, the dissociation constant of the mechanically reinforced states coincides with that measured for the low-avidity binding interface of protein L, suggesting a physiological role for the second binding interface. The molecular approach presented here opens the road to a new type of binding experiments, where the same molecule can be exposed to different solvents or ligands.

Climate change effects on tree growth from Romanian forest monitoring Level II plots.

Forest health status is negatively influenced by climate change, air pollution and other disturbances. Extreme droughts reduce stand productivity, increase vulnerability to pests, and can even provoke mortality. Growth dynamics at tree and forest stand levels are considered the main indicators of stability and productivity in forest ecosystem structures. The main climate drivers for tree growth were identified using basal area increment (BAI) as a synthetic indicator. BAI chronologies were obtained from increment cores for 1960-2012 period. Six species were analysed in an attempt to identify their growth limiting factors. For the most important oak species in Romania, resilience components were computed in order to analyse their response to drought events. Moreover, growth dynamics were analysed for two species in mixed and monoculture forests. The results suggest that - in comparison to Picea abies and Fagus sylvatica, the sensitivity of Quercus spp. is much higher (0.3-0.47). Oakspecies situated in the most drought-affected areas are sensitive to rainfall values from the previous autumn, current spring, and early summer, with April monthly values having the most significant effect on BAI increment (r = 0.47*) The most sensitive species to drought is Q. cerris and Q. frainetto. Their BAI reduction during drought is >50% compared with the BAI values before the drought period. The recovery capacity of tree growth following drought events is lower for Q. robur and Q. petraea and higher for Q. cerris and Q. frainetto. The mixed forest stands have not showed a constant higher resistance to drought.