Caspase-9 Is a Positive Regulator of Osteoblastic Cell Migration Identified by diaPASEF Proteomics.

Caspase-9 is traditionally considered the initiator caspase of the intrinsic apoptotic pathway. In the past decade, however, other functions beyond initiation/execution of cell death have been described including cell type-dependent regulation of proliferation, differentiation/maturation, mitochondrial, and endosomal/lysosomal homeostasis. As previous studies revealed nonapoptotic functions of caspases in osteogenesis and bone homeostasis, this study was performed to identify proteins and pathways deregulated by knockout of caspase-9 in mouse MC3T3-E1 osteoblasts. Data-independent acquisition-parallel accumulation serial fragmentation (diaPASEF) proteomics was used to compare protein profiles of control and caspase-9 knockout cells. A total of 7669 protein groups were quantified, and 283 upregulated/141 downregulated protein groups were associated with the caspase-9 knockout phenotype. The deregulated proteins were mainly enriched for those associated with cell migration and motility and DNA replication/repair. Altered migration was confirmed in MC3T3-E1 cells with the genetic and pharmacological inhibition of caspase-9. ABHD2, an established regulator of cell migration, was identified as a possible substrate of caspase-9. We conclude that caspase-9 acts as a modulator of osteoblastic MC3T3-E1 cell migration and, therefore, may be involved in bone remodeling and fracture repair.

Inhibition of caspase-8 cascade restrains the osteoclastogenic fate of bone marrow cells.

Osteoclasts are multinucleated cells of hematopoietic origin, with a pivotal role in bone development and remodeling. Failure in osteoclast differentiation and activation leads to various bone disorders; thus, attention has focused on a search of molecules involved in osteoclast regulatory pathways. Caspase-8 appears to be an interesting candidate for further exploration, due to its potential function in bone development and homeostasis. Mouse bone marrow cells were differentiated into osteoclasts by RANKL stimulation. Increased activation of caspase-8 and its downstream executioner caspases (caspase-3 and caspase-6) was found during osteoclastogenesis. Subsequent inhibition of caspase-8, caspase-3, or caspase-6, respectively, during osteoclast differentiation showed distinct changes in the formation of TRAP-positive multinucleated cells and reduced expression of osteoclast markers including Acp5, Ctsk, Dcstamp, and Mmp9. Analysis of bone matrix resorption confirmed significantly reduced osteoclast function after caspase inhibition. The results clearly showed the role of caspases in the proper development of osteoclasts and contributed new knowledge about non-apoptotic function of caspases.

Exploring caspase functions in mouse models.

Caspases are enzymes with protease activity. Despite being known for more than three decades, caspase investigation still yields surprising and fascinating information. Initially associated with cell death and inflammation, their functions have gradually been revealed to extend beyond, targeting pathways such as cell proliferation, migration, and differentiation. These processes are also associated with disease mechanisms, positioning caspases as potential targets for numerous pathologies including inflammatory, neurological, metabolic, or oncological conditions. While in vitro studies play a crucial role in elucidating molecular pathways, they lack the context of the body's complexity. Therefore, laboratory animals are an indispensable part of successfully understanding and applying caspase networks. This paper aims to summarize and discuss recent knowledge, understanding, and challenges in caspase knock-out mice.

DiaPASEF proteotype analysis indicates changes in cell growth and metabolic switch induced by caspase-9 inhibition in chondrogenic cells.

Caspase-9 is the major apical caspase responsible for triggering the intrinsic apoptotic pathway. Our previous study indicated that specific inhibition of caspase-9 caused microscopically evident alterations in appearance of the primary chondrogenic cultures which cannot be explained by decrease in apoptosis. To describe a complex molecular background of this effect, proteomics analysis of control and caspase-9 inhibitor-treated chondrogenic cultures were performed. Proteins were extracted, identified and quantified using LC-MS in both data dependent and data independent acquisition (DIA) mode. While directDIA analysis of diaPASEF data obtained using timsTOF Pro LC-MS system revealed 7849 protein groups (Q-value <0.01), a parallel analysis of iTRAQ-2DLC-MS3 and conventional DIA-MS data identified only 5146 and 4098 protein groups, respectively, showing diaPASEF a superior method for the study. The detailed analysis of diaPASEF data disclosed 236/551 significantly down-/up-regulated protein groups after caspase-9 inhibition, respectively (|log2FC|>0.58, Q value <0.05). Classification of downregulated proteins revealed changes in extracellular matrix organization, collagen metabolism, and muscle system processes. Moreover, deregulations suggest a switch from glycolytic to lipid based metabolism in the inhibited cells. No essential changes were found in the proteins involved in apoptosis. The data indicate new non-apoptotic participation of caspases in chondrocyte homeostasis with potential applications in cartilage pathophysiology.

Growth, physiology, and stomatal parameters of plant polyploids grown under ice age, present-day, and future CO2 concentrations.

Polyploidy plays an important role in plant evolution, but knowledge of its eco-physiological consequences, such as of the putatively enlarged stomata of polyploid plants, remains limited. Enlarged stomata should disadvantage polyploids at low CO2 concentrations (namely during the Quaternary glacial periods) because larger stomata are viewed as less effective at CO2 uptake. We observed the growth, physiology, and epidermal cell features of 15 diploids and their polyploid relatives cultivated under glacial, present-day, and potential future atmospheric CO2 concentrations (200, 400, and 800 ppm respectively). We demonstrated some well-known polyploidy effects, such as faster growth and larger leaves, seeds, stomata, and other epidermal cells. The stomata of polyploids, however, tended to be more elongated than those of diploids, and contrary to common belief, they had no negative effect on the CO2 uptake capacity of polyploids. Moreover, polyploids grew comparatively better than diploids even at low, glacial CO2 concentrations. Higher polyploids with large genomes also showed increased operational stomatal conductance and consequently, a lower water-use efficiency. Our results point to a possible decrease in growth superiority of polyploids over diploids in a current and future high CO2 climatic scenarios, as well as the possible water and/or nutrient dependency of higher polyploids.

A single-cell analytical approach to quantify activated caspase-3/7 during osteoblast proliferation, differentiation, and apoptosis.

The protein heterogeneity at the single-cell level has been recognized to be vital for an understanding of various life processes during animal development. In addition, the knowledge of accurate quantity of relevant proteins at cellular level is essential for appropriate interpretation of diagnostic and therapeutic results. Some low-copy-number proteins are known to play a crucial role during cell proliferation, differentiation, and also in apoptosis. The fate decision is often based on the concentration of these proteins in the individual cells. This is likely to apply also for caspases, cysteine proteases traditionally associated with cell death via apoptosis but recently being discovered also as important factors in cell proliferation and differentiation. The hypothesis was tested in bone-related cells, where modulation of fate from apoptosis to proliferation/differentiation and vice versa is particularly challenging, e.g., towards anti-osteoporotic treatments and anti-cancer strategies. An ultrasensitive and highly selective method based on bioluminescence photon counting was used to quantify activated caspase-3/7 in order to demonstrate protein-level heterogeneity in individual cells within one population and to associate quantitative measurements with different cell fates (proliferation, differentiation, apoptosis). The results indicate a gradual increase of caspase-3/7 activation from the proliferative status to differentiation (more than three times) and towards apoptosis (more than six times). The findings clearly support one of the putative key mechanisms of non-apoptotic functions of pro-apoptotic caspases based on fine-tuning of their activation levels.

Caspase-1 Inhibition Impacts the Formation of Chondrogenic Nodules, and the Expression of Markers Related to Osteogenic Differentiation and Lipid Metabolism.

Caspase-1, as the main pro-inflammatory cysteine protease, was investigated mostly with respect to inflammation-related processes. Interestingly, caspase-1 was identified as being involved in lipid metabolism, which is extremely important for the proper differentiation of chondrocytes. Based on a screening investigation, general caspase inhibition impacts the expression of Cd36 in chondrocytes, the fatty acid translocase with a significant impact on lipid metabolism. However, the engagement of individual caspases in the effect has not yet been identified. Therefore, the hypothesis that caspase-1 might be a candidate here appears challenging. The primary aim of this study thus was to find out whether the inhibition of caspase-1 activity would affect Cd36 expression in a chondrogenic micromass model. The expression of Pparg, a regulator Cd36, was examined as well. In the caspase-1 inhibited samples, both molecules were significantly downregulated. Notably, in the treated group, the formation of the chondrogenic nodules was apparently disrupted, and the subcellular deposition of lipids and polysaccharides showed an abnormal pattern. To further investigate this observation, the samples were subjected to an osteogenic PCR array containing selected markers related to cartilage/bone cell differentiation. Among affected molecules, Bmp7 and Gdf10 showed a significantly increased expression, while Itgam, Mmp9, Vdr, and Rankl decreased. Notably, Rankl is a key marker in bone remodeling/homeostasis and thus is a target in several treatment strategies, including a variety of fatty acids, and is balanced by its decoy receptor Opg (osteoprotegerin). To evaluate the effect of Cd36 downregulation on Rankl and Opg, Cd36 silencing was performed using micromass cultures. After Cd36 silencing, the expression of Rankl was downregulated and Opg upregulated, which was an inverse effect to caspase-1 inhibition (and Cd36 upregulation). These results demonstrate new functions of caspase-1 in chondrocyte differentiation and lipid metabolism-related pathways. The effect on the Rankl/Opg ratio, critical for bone maintenance and pathology, including osteoarthritis, is particularly important here as well.

Spatial and Temporal Variability of Plant Leaf Responses Cascade after PSII Inhibition: Raman, Chlorophyll Fluorescence and Infrared Thermal Imaging.

The use of photosystem II (PSII) inhibitors allows simulating cascade of defense and damage responses, including the oxidative stress. In our study, PSII inhibiting herbicide metribuzin was applied to the leaf of the model plant species Chenopodium album. The temporally and spatially resolved cascade of defense responses was studied noninvasively at the leaf level by combining three imaging approaches: Raman spectroscopy as a principal method, corroborated by chlorophyll a fluorescence (ChlF) and infrared thermal imaging. ChlF imaging show time-dependent transport in acropetal direction through veins and increase of area affected by metribuzin and demonstrated the ability to distinguish between fast processes at the level of electron transport (1 - Vj) from slow processes at the level of non-photochemical energy dissipation (NPQ) or maximum efficiency of PSII photochemistry (Fv/Fm). The high-resolution resonance Raman images show zones of local increase of carotenoid signal 72 h after the herbicide application, surrounding the damaged tissue, which points to the activation of defense mechanisms. The shift in the carotenoid band indicates structural changes in carotenoids. Finally, the increase of leaf temperature in the region surrounding the spot of herbicide application and expanding in the direction to the leaf tip proves the metribuzin effect on slow stomata closure.

Growth-dependent phenotype in FasL-deficient mandibular/alveolar bone.

FASL (CD178) is known for its role in triggering apoptosis, mostly in relation with immune cells but additional functions have been reported more recently, including those in bone development. Examination of postnatal FasL-deficient mice (gld) showed an increased bone deposition in adult mice when compared with wild types. However, a different phenotype was observed prenatally, when the gld bone was underdeveloped. The aim of the following investigation was to evaluate this indication for an growth-dependent bone phenotype of gld mice and to search for the 'switch point'. This study focused on the mandibular/alveolar bone as an important structure for tooth anchorage. In vivo micro-computed tomography (CT) analysis was performed at different stages during the first month (6, 12 and 24 days) of postnatal bone development. In 6-day-old gld mice, a decrease in bone volume/tissue volume (BV/TV), trabecular thickness and trabecular number was revealed. In contrast, the 12-day-old gld mice showed an increased BV/TV and trabecular thickness in the alveolar bone. The same observation applied for bone status in 24-day-old gld mice. Therefore, changes in the bone phenotype occurred between day 6 and 12 of the postnatal development. The switch point is likely related to the changing proportion of bone cells at these stages of development, when the number of osteocytes increases. Indeed, the immunohistochemical analysis of FASL localized this protein in osteoblasts, whereas osteocytes were mostly negative at examined stages. The impact of FASL particularly on osteoblasts would agree with an earlier in vivo observed effect of FASL deficiency on expression of Mmp2, typical for osteoblasts, in the gld mandibular/alveolar bone. Notably, an age-dependent bone phenotype was reported in Mmp2-deficient mice.

Osteogenic Profile of Mesenchymal Cell Populations Contributing to Alveolar Bone Formation.

Teeth develop within the surrounding periodontal tissues, involving the alveolar bone, periodontal ligament and cementum. The alveolar bone originates through the process of intramembranous ossification involving mesenchymal cells from the tooth germ. As most available data are related to endochondral ossification, we examined the molecular background of alveolar bone development. We investigated the osteogenic profile of mesenchymal cells dissected from mouse mandible slices at the stage of early alveolar bone formation. Relative monitoring of gene expression was undertaken using PCR Arrays; this included the profiles of 84 genes associated with osteogenesis. To examine the tooth-bone interface, stages with detectable changes in bone remodelling during development (E13.0, E14.0 and E15.0) were chosen and compared with each other. These results showed a statistically significant increase in the expression of the genes Fgf3, Ctsk, Icam-1, Mmp9, Itga3 and Tuft1, and of a wide range of collagens (Col1a2, Col3a1, Col7a1, Col12a1, Col14a1). Decreased expression was detected in the case of Col2a1, Sox9, Smad2 and Vegfb. To confirm these changes in gene expression, immunofluorescence analyses of Mmp9 and Sox9 proteins were performed in situ. Our research has identified several candidate genes that may be crucial for the initiation of alveolar bone formation and is the basis for further functional studies.

Expression of apoptosis-related genes in the mouse skin during the first postnatal catagen stage, focused on localization of Bnip3L and caspase-12.

Hair follicles undergo repetitive stages of cell proliferation and programmed cell death. The catagen stage of physiological apoptosis is connected with dynamic changes in morphology and alterations in gene expression. However, hair follicle apoptosis must be in balance with events in surrounding tissues, such as keratinocyte cornification, to maintain complex skin homeostasis. Several pro- and anti-apoptotic molecules in the skin have been reported but mainly in pathological states. In this investigation, apoptosis-related gene expression was examined during the first catagen stage of mouse hair follicle development by PCR arrays under physiological conditions. Postnatal stages P15 and P17, representing early and late catagen stages, were evaluated relatively to stage P6, representing the hair follicle growing phase, to demonstrate dynamics of gene activation during the catagen. Several statistically significant alterations were observed at P15 and particularly at P17. Bnip3L and caspase-12 identified by the PCR arrays at both catagen stages were additionally localized using immunofluorescence and were reported in physiological hair development for the first time.

Embryonic toxicity of nanoparticles.

Applications of nanoparticles (NP) in medicine, industry and other branches of human activities undoubtedly contribute to technology development and well-being. However, as NP are very small units in a wide range of materials, there is a lack of information related to possible side effects potentially affecting the health of organisms. There is increasing experimental interest in the determination of environmental effects on humans exposed to NP. Most such experimental studies focus on adult populations or adult experimental animals. However, embryos can be more sensitive to pollutants and environmental impacts in some species. Therefore, some investigations dealing particularly with the effects of NP on embryonic development have appeared recently and this issue is becoming of great concern. The aim of this review is to summarize the knowledge on the effects of various nanomaterials on embryonic development. A comprehensive collection of significant experimental nanotoxicity data is presented, which also indicate how the toxic effect of NP can be mediated and modulated with respect to possible effective protection strategies.

Inhibition of caspase-11 under inflammatory conditions suppresses chondrogenic differentiation.

Caspase-11 is the murine homologue of human caspases-4 and -5 and is involved in mediating the inflammatory response. However, its functions are often confused and misinterpreted with the more important and better described caspase-1. Therefore, this study focused exclusively on the specific roles of caspase-11, both in cartilage formation and in the inflammatory environment. The presence of caspase-11 during mouse limb development and in chondrogenic cell cultures was investigated by immunofluorescence detection. Subsequently, the function of caspase-11 was downregulated and the affected molecules investigated. The expression analysis applied for osteo/chondrogenesis associated factors and inflammatory cytokines. Simultaneously, morphological appearance of the micromass cultures was evaluated. The results revealed that caspase-11 is physiologically present during cartilage development, but its inhibition under physiological conditions has no significant effect on chondrogenic differentiation. However, in an inflammatory environment, inhibition and downregulation of caspase-11 leads to reduced differentiation of cartilage nodules. Additionally, reduced expression of several genes including Col2a1 and Sp7 and conversely increased expression of Mmp9 were observed. In the cytokine expression panel, a significant decrease was found in molecules that, along with the inflammatory function, may also be involved in cartilage differentiation. The findings bring new information about caspase-11 in chondrogenesis and show that its downregulation under inflammatory conditions reduces cartilage formation.

The effect of elevated CO2 on photosynthesis is modulated by nitrogen supply and reduced water availability in Picea abies.

It is assumed that the stimulatory effects of elevated CO2 concentration ([CO2]) on photosynthesis and growth may be substantially reduced by co-occurring environmental factors and the length of CO2 treatment. Here, we present the study exploring the interactive effects of three manipulated factors ([CO2], nitrogen supply and water availability) on physiological (gas-exchange and chlorophyll fluorescence), morphological and stoichiometric traits of Norway spruce (Picea abies) saplings after 2 and 3 years of the treatment under natural field conditions. Such multifactorial studies, going beyond two-way interactions, have received only limited attention until now. Our findings imply a significant reduction of [CO2]-enhanced rate of CO2 assimilation under reduced water availability which deepens with the severity of water depletion. Similarly, insufficient nitrogen availability leads to a down-regulation of photosynthesis under elevated [CO2] being particularly associated with reduced carboxylation efficiency of the Rubisco enzyme. Such adjustments in the photosynthesis machinery result in the stimulation of water-use efficiency under elevated [CO2] only when it is combined with a high nitrogen supply and reduced water availability. These findings indicate limited effects of elevated [CO2] on carbon uptake in temperate coniferous forests when combined with naturally low nitrogen availability and intensifying droughts during the summer periods. Such interactions have to be incorporated into the mechanistic models predicting changes in terrestrial carbon sequestration and forest growth in the future.

Markers of dental pulp stem cells in in vivo developmental context.

Teeth and their associated tissues contain several populations of mesenchymal stem cells, one of which is represented by dental pulp stem cells (DPSCs). These cells have mainly been characterised in vitro and numerous positive and negati ve markers for these cells have been suggested. To investigate the presence and localization of these molecules during development, forming dental pulp was examined using the mouse first mandibular molar as a model. The stages corresponding to postnatal (P) days 0, 7, 14, and 21 were investigated. The expression was monitored using customised PCR Arrays. Additionally, in situ localization of the key trio of markers (Cd73, Cd90, Cd105 coded by genes Nt5e, Thy1, Eng) was performed at prenatal and postnatal stages using immunohistochemistry. The expression panel of 24 genes assigned as in vitro markers of DPSCs or mesenchymal stem cells (MSCs) revealed their developmental dynamics during formation of dental pulp mesenchyme. Among the positive markers, Vcam1, Fgf2, Nes were identified as increasing and Cd44, Cd59b, Mcam, Alcam as decreasing between perinatal vs. postnatal stages towards adulthood. Within the panel of negative DPSC markers, Cd14, Itgb2, Ptprc displayed increased and Cd24a decreased levels at later stages of pulp formation. Within the key trio of markers, Nt5e did not show any significant expression difference within the investigated period. Thy1 displayed a strong decrease between P0 and P7 while Eng increased between these stages. In situ localization of Cd73, Cd90 and Cd105 showed them overlap in differentiated odontoblasts and in the sub-odontoblastic layer that is speculated to host odontoblast progenitors. The highly prevalent expression of particularly Cd73 and Cd90 opens the question of potential multiple functions of these molecules. The results from this study add to the in vitro based knowledge by showing dynamics in the expression of DPSC/MSC markers during dental pulp formation in an in vivo context and thus with respect to the natural environment important for commitment of stem cells.

Interactive effects of UV radiation and water deficit on production characteristics in upland grassland and their estimation by proximity sensing.

An increase in extreme weather and changes in other conditions associated with ongoing climate change are exposing ecosystems to a very wide range of environmental drivers that interact in ways which are not sufficiently understood. Such uncertainties in how ecosystems respond to multifactorial change make it difficult to predict the impacts of environmental change on ecosystems and their functions. Since water deficit (WD) and ultraviolet radiation (UV) trigger similar protective mechanisms in plants, we tested the hypothesis that UV modulates grassland acclimation to WD, mainly through changes in the root/shoot (R/S) ratio, and thus enhances the ability of grassland to acquire water from the soil and hence maintain its productivity. We also tested the potential of spectral reflectance and thermal imaging for monitoring the impacts of WD and UV on grassland production parameters. The experimental plots were manipulated by lamellar shelters allowing precipitation to pass through or to be excluded. The lamellas were either transmitting or blocking the UV. The results show that WD resulted in a significant decrease in aboveground biomass (AB). In contrast, belowground biomass (BB), R/S ratio, and total biomass (TB) increased significantly in response to WD, especially in UV exclusion treatment. UV exposure had a significant effect on AB and BB, but only in the last year of the experiment. The differences in the effect of WD between years show that the effect of precipitation removal is largely influenced by the potential evapotranspiration (PET) in a given year and hence mainly by air temperatures, while the resulting effect on production parameters is best correlated with the water balance given by the difference between precipitation and PET. Canopy temperature and selected spectral reflectance indices showed a significant response to WD and also significant relationships with morphological (AB, R/S) and biochemical (C/N ratio) parameters. In particular, the vegetation indices NDVI and RDVI provided the best correlations of biomass changes caused by WD and thus the highest potential to remotely sense drought effects on terrestrial vegetation.

Autophagy-related proteases accompany the transition of pre-chondrogenic cells into chondroblasts.

BACKGROUND: Autophagy is classified as a form of programmed cell death. Nevertheless, besides the death-inducing function, autophagy enables removal of damaged organelles, energy savings, and thus cell survival. This applies in particular to cells with poor renewal capabilities, such as chondroblasts. Autophagy is regulated by a complex molecular network, including proteases and their substrates. In autopodium, autophagy-related proteases have been examined particularly within the context of the elimination of the interdigital tissue. However, the death-inducing effects of their expression/activation have not been specified yet. This work focuses on autophagy-associated proteases (cathepsins, matrix metalloproteinases, and caspases) in development of phalangeal cartilage of the mouse autopodium. METHODS: PCR Array, Real-time PCR, and immunohistochemistry were used to follow the expression of autophagy-associated genes in vivo at two developmental stages prenatal/embryonic (E)12 vs. E14. Real-time PCR was then applied to investigate the influence of rapamycin (an inducer of autophagy) on the expression of autophagy-associated proteases in chondroblasts in vitro using micromass culture. RESULTS: Several proteases showed increased expression levels during the transition of pre-chondrogenic cells into chondroblasts in vivo. The most significant increases were observed for Ctsb (fold regulation 2.22), Ctsd (fold regulation 2.37), Ctss (fold regulation 2.92), Mmp9 (up to 445%), and Casp8 (up to 250%). The transition was associated also with the high expression of crucial autophagic inducers, such as Atgs. The in vitro treatment of chondroblasts by rapamycin showed significantly decreased expression of cathepsins, a mild increase in expression of metalloproteinases, and no effect in caspase expression. CONCLUSIONS: The present data provide a screening of autophagy-associated proteases accompanying the formation of cartilage in vivo and specify their expression under rapamycin treatment in vitro. Notably, the selected proteases are assigned to osteoarthritis, therefore their regulation might be used in clinically oriented studies.

Caspase-8 Deficient Osteoblastic Cells Display Alterations in Non-Apoptotic Pathways.

Caspase-8 is the key component of the receptor-mediated (extrinsic) apoptotic pathway. Immunological localization of active caspase-8 showed its presence in osteoblasts, including non-apoptotic ones. Further in vivo exploration of caspase-8 functions in the bone is hindered by the fact that the caspase-8 knock-out is lethal prenatally. Examinations were thus performed using individual cell populations in vitro. In this study, caspase-8 was eliminated by the CRISPR/cas9 technology in MC3T3-E1 cells, the most common in vitro model of osteoblastic populations. The aim of the work was to specify the consequences of caspase-8 deficiency on non-apoptotic pathways. The impact on the osteogenic gene expression of the osteoblastic cells along with alterations in proliferation, caspase cascades and rapamycin induced autophagy response were evaluated. Osteogenic differentiation of caspase-8 deficient cells was inhibited as these cells displayed a decreased level of mineralization and lower activity of alkaline phosphatase. Among affected osteogenic genes, based on the PCR Array, major changes were observed for Ctsk, as down-regulated, and Gdf10, as up-regulated. Other significantly down-regulated genes included those coding osteocalcin, bone morphogenetic proteins (-3, -4 and -7), collagens (-1a1, -14a1) or Phex. The formation of autophagosomes was not altered in rapamycin-treated caspase-8 deficient cells, but expression of some autophagy-related genes, including Tnfsf10, Cxcr4, Dapk1 and Igf1, was significantly downregulated. These data provide new insight into the effects of caspase-8 on non-apoptotic osteogenic pathways.

UV radiation and drought interact differently in grass and forb species of a mountain grassland.

Among abiotic stressors, drought and enhanced ultraviolet radiation (UV) received a lot of attention, because of their potential to impair plant growth. Since drought and UV induce partially similar protective mechanisms, we tested the hypothesis that UV ameliorates the effect of reduced water availability (WA) in selected grass (Holcus mollis and Agrostis capillaris) and forb species (Hypericum maculatum and Rumex acetosa). During 2011-2014, an outdoor manipulation experiment was conducted on a mountain grassland ecosystem (Beskydy Mts; Czech Republic). Lamellar shelters were used to pass (WAamb) or exclude (WA-) incident precipitation in order to simulate reduced water availability (WA). In addition, the lamellas were made from acrylics either transmitting (UVamb) or blocking (UV-) incident UV. Generally, both UV exposure and reduced WA enhanced epidermal UV-screening, while exposure to both factors resulted in less than additive interactions. Although UV radiation increased epidermal UV-screening rather in the grass (up to 29 % in A. capillaris) than forb (up to 12 % in H. maculatum) species and rather in well-watered than reduced WA plants, such acclimation response did not result in significant alleviation of reduced WA effects on gas exchange and morphological parameters. The study contributes to a better understanding of plant responses to complex environmental conditions and will help for successful modelling forecasts of future climate change impacts.

Single and interactive effects of variables associated with climate change on wheat metabolome.

One of the key challenges linked with future food and nutritional security is to evaluate the interactive effect of climate variables on plants' growth, fitness, and yield parameters. These interactions may lead to unique shifts in the morphological, physiological, gene expression, or metabolite accumulation patterns, leading to an adaptation response that is specific to future climate scenarios. To understand such changes, we exposed spring wheat to 7 regimes (3 single and 4 combined climate treatments) composed of elevated temperature, the enhanced concentration of CO2, and progressive drought stress corresponding to the predicted climate of the year 2100. The physiological and metabolic responses were then compared with the current climate represented by the year 2020. We found that the elevated CO2 (eC) mitigated some of the effects of elevated temperature (eT) on physiological performance and metabolism. The metabolite profiling of leaves revealed 44 key metabolites, including saccharides, amino acids, and phenolics, accumulating contrastingly under individual regimes. These metabolites belong to the central metabolic pathways that are essential for cellular energy, production of biosynthetic pathways precursors, and oxidative balance. The interaction of eC alleviated the negative effect of eT possibly by maintaining the rate of carbon fixation and accumulation of key metabolites and intermediates linked with the Krebs cycle and synthesis of phenolics. Our study for the first time revealed the influence of a specific climate factor on the accumulation of metabolic compounds in wheat. The current work could assist in the understanding and development of climate resilient wheat by utilizing the identified metabolites as breeding targets for food and nutritional security.