Responses to Airborne Ozone and Soilborne Metal Pollution in Afforestation Plants with Different Life Forms.

With the current increases in environmental stress, understanding species-specific responses to multiple stress agents is needed. This science is especially important for managing ecosystems that are already confronted with considerable pollution. In this study, responses to ozone (O3, ambient daily course values + 20 ppb) and mixed metal contamination in soils (MC, cadmium/copper/lead/zinc = 25/1100/2500/1600 mg kg-1), separately and in combination, were evaluated for three plant species (Picea abies, Acer pseudoplatanus, Tanacetum vulgare) with different life forms and ecological strategies. The two treatments elicited similar stress reactions, as shown by leaf functional traits, gas exchange, tannin, and nutrient markers, irrespective of the plant species and life form, whereas the reactions to the treatments differed in magnitude. Visible and microscopic injuries at the organ or cell level appeared along the penetration route of ozone and metal contamination. At the whole plant level, the MC treatment caused more severe injuries than the O3 treatment and few interactions were observed between the two stress factors. Picea trees, with a slow-return strategy, showed the highest stress tolerance in apparent relation to an enhancement of conservative traits and an exclusion of stress agents. The ruderal and more acquisitive Tanacetum forbs translocated large amounts of contaminants above ground, which may be of concern in a phytostabilisation context. The deciduous Acer trees-also with an acquisitive strategy-were most sensitive to both stress factors. Hence, species with slow-return strategies may be of particular interest for managing metal-polluted sites in the current context of multiple stressors and for safely confining soil contaminants below ground.

Ante- and post-mortem cellular injury dynamics in hybrid poplar foliage as a function of phytotoxic O3 dose.

After reaching phytotoxic levels during the last century, tropospheric ozone (O3) pollution is likely to remain a major concern in the coming decades. Despite similar injury processes, there is astounding interspecific-and sometimes intraspecific-foliar symptom variability, which may be related to spatial and temporal variation in injury dynamics. After characterizing the dynamics of physiological responses and O3 injury in the foliage of hybrid poplar in an earlier study, here we investigated the dynamics of changes in the cell structure occurring in the mesophyll as a function of O3 treatment, time, phytotoxic O3 dose (POD0), leaf developmental stage, and mesophyll layer. While the number of Hypersensitive Response-like (HR-like) lesions increased with higher O3 concentrations and POD0, especially in older leaves, most structural HR-like markers developed after cell death, independent of the experimental factors. The pace of degenerative Accelerated Cell Senescence (ACS) responses depended closely on the O3 concentration and POD0, in interaction with leaf age. Changes in total chlorophyll content, plastoglobuli and chloroplast shape pointed to thylakoid membranes in chloroplasts as being especially sensitive to O3 stress. Hence, our study demonstrates that early HR-like markers can provide reasonably specific, sensitive and reliable quantitative structural estimates of O3 stress for e.g. risk assessment studies, especially if they are associated with degenerative and thylakoid-related injury in chloroplasts from mesophyll.

Metal Accumulation and Biomass Production in Young Afforestations Established on Soil Contaminated by Heavy Metals.

The restoration of forest ecosystems on metal-contaminated sites can be achieved whilst producing valuable plant biomass. Here, we investigated the metal accumulation and biomass production of young afforestations on contaminated plots by simulating brownfield site conditions. On 16 3-m2 plots, the 15 cm topsoil was experimentally contaminated with Zn/Cu/Pb/Cd = 2854/588/103/9.2 mg kg-1 using smelter filter dust, while 16 uncontaminated plots (Zn/Cu/Pb/Cd = 97/28/37/< 1) were used as controls. Both the calcareous (pH 7.4) and acidic (pH 4.2) subsoils remained uncontaminated. The afforestations consisted of groups of conifers, deciduous trees, and understorey plants. During the four years of cultivation, 2254/86/0.35/10 mg m-2 Zn/Cu/Pb/Cd were extracted from the contaminated soils and transferred to the aboveground parts of the plants (1279/72/0.06/5.5 mg m-2 in the controls). These extractions represented 3/2/3% of the soluble soil Zn/Cu/Cd fractions. The conifers showed 4-8 times lower root-to-shoot translocation of Cu and Zn than the deciduous trees. The contamination did not affect the biomass of the understorey plants and reduced that of the trees by 23% at most. Hence, we conclude that the afforestation of brown field sites with local tree species is an interesting option for their reclamation from an ecological as well as economic perspective.

Dynamics of Foliar Responses to O3 Stress as a Function of Phytotoxic O3 Dose in Hybrid Poplar.

With background concentrations having reached phytotoxic levels during the last century, tropospheric ozone (O3) has become a key climate change agent, counteracting carbon sequestration by forest ecosystems. One of the main knowledge gaps for implementing the recent O3 flux-based critical levels (CLs) concerns the assessment of effective O3 dose leading to adverse effects in plants. In this study, we investigate the dynamics of physiological, structural, and morphological responses induced by two levels of O3 exposure (80 and 100 ppb) in the foliage of hybrid poplar, as a function of phytotoxic O3 dose (POD0) and foliar developmental stage. After a latency period driven by foliar ontological development, the gas exchanges and chlorophyll content decreased with higher POD0 monotonically. Hypersensitive response-like lesions appeared early during exposure and showed sigmoidal-like dynamics, varying according to leaf age. At current POD1_SPEC CL, notwithstanding the aforementioned reactions and initial visible injury to foliage, the treated poplars had still not shown any growth or biomass reduction. Hence, this study demonstrates the development of a complex syndrome of early reactions below the flux-based CL, with response dynamics closely determined by the foliar ontological stage and environmental conditions. General agreement with patterns observed in the field appears indicative of early O3 impacts on processes relevant, e.g., biodiversity ecosystem services before those of economic significance - i.e., wood production, as targeted by flux-based CL.

Salt accumulation and effects within foliage of Tilia × vulgaris trees from the street greenery of Riga, Latvia.

Green infrastructures within sprawling cities provide essential ecosystem services, increasingly undermined by environmental stress. The main objective in this study was to relate the allocation patterns of NaCl contaminants to injury within foliage of lime trees mechanistically and distinguish between the effects of salt and other environmental stressors. Using field material representative of salt contamination levels in the street greenery of Riga, Latvia, the contribution of salt contaminants to structural and ultrastructural injury was analyzed, combining different microscopy techniques. On severely salt-polluted and dystrophic soils, the foliage of street lime trees showed foliar concentrations of Na/Cl up to 13,600/16,750 mg kg-1 but a still balanced nutrient content. The salt contaminants were allocated to all leaf blade tissues and accumulated in priority within mesophyll vacuoles, changing the vacuolar ionic composition at the expense of especially K and Ca. The size of mesophyll cells and vacuoles was increased as a function of NaCl concentration, suggesting impeded transpiration stream. In parallel, the cytoplasm showed degenerative changes, suggesting indirect stress effects. Hence, the lime trees in Riga showed tolerance to the dystrophic environmental conditions enhanced by salt pollution but their leaf physiology appeared directly impacted by the accumulation of contaminants within foliage.

How does varying water supply affect oxygen isotope variations in needles and tree rings of Scots pine?

In many regions, drought is suspected to be a cause of Scots pine decline and mortality, but the underlying physiological mechanisms remain unclear. Because of their relationship to ecohydrological processes, δ18O values in tree rings are potentially useful for deciphering long-term physiological responses and tree adaptation to increasing drought. We therefore analyzed both needle- and stem-level isotope fractionations in mature trees exposed to varying water supply. In a first experiment, we investigated seasonal δ18O variations in soil and needle water of Scots pine in a dry inner Alpine valley in Switzerland, comparing drought-stressed trees with trees that were irrigated for more than 10 years. In a second experiment, we analyzed twentieth-century δ18O variations in tree rings of the same forest, including a group of trees that had recently died. We observed less 18O enrichment in needle water of drought-stressed compared with irrigated trees. We applied different isotope fractionation models to explain these results, including the Péclet and the two-pool correction, which considers the ratio of unenriched xylem water in the needles to total needle water. Based on anatomical measurements, we found this ratio to be unchanged in drought-stressed needles, although they were shorter. The observed lower 18O enrichment in needles of stressed trees was therefore likely caused by increased effective path length for water movement within the leaf lamina. In the tree-ring study, we observed lower δ18O values in tree rings of dead trees compared with survivors during several decades prior to their death. These lower values in declining trees are consistent with the lower needle water 18O enrichment observed for drought-stressed compared with irrigated trees, suggesting that this needle-level signal is reflected in the tree rings, although changes in rooting depth could also play a role. Our study demonstrates that long-term effects of drought are reflected in the tree-ring δ18O values, which helps to provide a better understanding of past tree physiological changes of Scots pine.

Transmembrane transport and stress response genes play an important role in adaptation of Arabidopsis halleri to metalliferous soils.

When plants adapt to local environments, strong signatures of selection are expected in the genome, particularly in high-stress environments such as trace metal element enriched (metalliferous) soils. Using Arabidopsis halleri, a model species for metal homeostasis and adaptation to extreme environments, we identifid genes, gene variants, and pathways that are associated with soil properties and may thus contribute to adaptation to high concentrations of trace metal elements. We analysed whole-genome Pool-seq data from two metallicolous (from metalliferous soils) and two non-metallicolous populations (in total 119 individuals) and associated allele frequencies of the identified single-nucleotide polymorphisms (SNPs) with soil variables measured on site. Additionally, we accounted for polygenic adaptation by searching for gene pathways showing enrichment of signatures of selection. Out of >2.5 million SNPs, we identified 57 SNPs in 19 genes that were significantly associated with soil variables and are members of three enriched pathways. At least three of these candidate genes and pathways are involved in transmembrane transport and/or associated with responses to various stresses such as oxidative stress. We conclude that both allocation and detoxification processes play a crucial role in A. halleri for coping with these unfavourable conditions.

Ozone affects leaf physiology and causes injury to foliage of native tree species from the tropical Atlantic Forest of southern Brazil.

In southern Brazil, the recent increase in tropospheric ozone (O3) concentrations poses an additional threat to the biodiverse but endangered and fragmented remnants of the Atlantic Forest. Given the mostly unknown sensitivity of tropical species to oxidative stress, the principal objective of this study was to determine whether the current O3 levels in the Metropolitan Region of Campinas (MRC), downwind of São Paulo, affect the native vegetation of forest remnants. Foliar responses to O3 of three tree species typical of the MRC forests were investigated using indoor chamber exposure experiments under controlled conditions and a field survey. Exposure to 70ppb O3 reduced assimilation and leaf conductance but increased respiration in Astronium graveolens while gas exchange in Croton floribundus was little affected. Both A. graveolens and Piptadenia gonoacantha developed characteristic O3-induced injury in the foliage, similar to visible symptoms observed in >30% of trees assessed in the MRC, while C. floribundus remained asymptomatic. The underlying structural symptoms in both O3-exposed and field samples were indicative of oxidative burst, hypersensitive responses, accelerated cell senescence and, primarily in field samples, interaction with photo-oxidative stress. The markers of O3 stress were thus mostly similar to those observed in other regions of the world. Further research is needed, to estimate the proportion of sensitive forest species, the O3 impact on tree growth and stand stability and to detect O3 hot spots where woody species in the Atlantic Forest are mostly affected.

Responses of beech and spruce foliage to elevated carbon dioxide, increased nitrogen deposition and soil type.

Although enhanced carbon fixation by forest trees may contribute significantly to mitigating an increase in atmospheric carbon dioxide (CO2), capacities for this vary greatly among different tree species and locations. This study compared reactions in the foliage of a deciduous and a coniferous tree species (important central European trees, beech and spruce) to an elevated supply of CO2 and evaluated the importance of the soil type and increased nitrogen deposition on foliar nutrient concentrations and cellular stress reactions. During a period of 4 years, beech (represented by trees from four different regions) and spruce saplings (eight regions), planted together on either acidic or calcareous forest soil in the experimental model ecosystem chambers, were exposed to single and combined treatments consisting of elevated carbon dioxide (+CO2, 590 versus 374 µL L(-1)) and elevated wet nitrogen deposition (+ND, 50 versus 5 kg ha(-1) a(-1)). Leaf size and foliage mass of spruce were increased by +CO2 on both soil types, but those of beech by +ND on the calcareous soil only. The magnitude of the effects varied among the tree origins in both species. Moreover, the concentration of secondary compounds (proanthocyanidins) and the leaf mass per area, as a consequence of cell wall thickening, were also increased and formed important carbon sinks within the foliage. Although the species elemental concentrations differed in their response to CO2 fertilization, the +CO2 treatment effect was weakened by an acceleration of cell senescence in both species, as shown by a decrease in photosynthetic pigment and nitrogen concentration, discolouration and stress symptoms at the cell level; the latter were stronger in beech than spruce. Hence, young trees belonging to a species with different ecological niches can show contrasting responses in their foliage size, but similar responses at the cell level, upon exposure to elevated levels of CO2. The soil type and its nutrient supply largely determined the fertilization gain, especially in the case of beech trees with a narrow ecological amplitude.

Ozone phytotoxic potential with regard to fragments of the Atlantic Semi-deciduous Forest downwind of Sao Paulo, Brazil.

In the Metropolitan Region of Campinas (MRC), Brazil, high levels of primary pollutants contribute to ozone (O3) formation. However, little is known regarding the O3 effects in the tropics. Objectives in this study were to characterize the present levels of O3 pollution and to evaluate the relevance of current concentration-based indices for assessing the phytotoxic potential of O3. Changes in O3 concentrations and precursors at 5 monitoring stations within towns of MRC were analyzed. The daily O3 profile was typical for urban sites and showed little yearly variation. Given the permanently foliated forest canopy, yearly rather than seasonal O3 indices were thus more appropriate for estimating the effective ozone dose. With yearly SUM00, SUM60 and AOT40 of 156, 16 and 14 ppm h and confirmed by evidence of O3 injury in foliage, oxidative stress in the MRC has reached levels high enough to affect trees from the Atlantic Semi-deciduous Forest.

Foliar symptoms triggered by ozone stress in irrigated holm oaks from the city of Madrid, Spain.

BACKGROUND: Despite abatement programs of precursors implemented in many industrialized countries, ozone remains the principal air pollutant throughout the northern hemisphere with background concentrations increasing as a consequence of economic development in former or still emerging countries and present climate change. Some of the highest ozone concentrations are measured in regions with a Mediterranean climate but the effect on the natural vegetation is alleviated by low stomatal uptake and frequent leaf xeromorphy in response to summer drought episodes characteristic of this climate. However, there is a lack of understanding of the respective role of the foliage physiology and leaf xeromorphy on the mechanistic effects of ozone in Mediterranean species. Particularly, evidence about morphological and structural changes in evergreens in response to ozone stress is missing. RESULTS: Our study was started after observing ozone -like injury in foliage of holm oak during the assessment of air pollution mitigation by urban trees throughout the Madrid conurbation. Our objectives were to confirm the diagnosis, investigate the extent of symptoms and analyze the ecological factors contributing to ozone injury, particularly, the site water supply. Symptoms consisted of adaxial and intercostal stippling increasing with leaf age. Underlying stippling, cells in the upper mesophyll showed HR-like reactions typical of ozone stress. The surrounding cells showed further oxidative stress markers. These morphological and micromorphological markers of ozone stress were similar to those recorded in deciduous broadleaved species. However, stippling became obvious already at an AOT40 of 21 ppm•h and was primarily found at irrigated sites. Subsequent analyses showed that irrigated trees had their stomatal conductance increased and leaf life -span reduced whereas the leaf xeromorphy remained unchanged. These findings suggest a central role of water availability versus leaf xeromorphy for ozone symptom expression by cell injury in holm oak.

Concomitant dendrometer and leaf patch pressure probe measurements reveal the effect of microclimate and soil moisture on diurnal stem water and leaf turgor variations in young oak trees.

Tree water relations and their dependence on microclimate and soil moisture were studied over several months in young oaks (Quercus robur L.) subjected in large lysimeter-based open top chambers to environments with a controlled soil water supply. Automated single point dendrometers and the recently developed leaf patch clamp pressure (LPCP) probe were used for monitoring water-related stem radius variations (ΔW) and turgor-dependent leaf patch pressures (Pp). Both parameters showed distinct diurnal patterns with sharp negative and positive peaking of ΔW and Pp, respectively, after solar noon and recovery to initial levels in the evening. During the day, varying solar radiation was responsible for short time fluctuations of Pp in the range of minutes to hours reflecting feedback regulation of leaf turgor by sunlight driven stomatal movements. At longer timescales, i.e. days to months, atmospheric vapour pressure deficit (VPD) and soil water content (SWC) were the main determinants of ΔW and Pp. Daily minimum and maximum values of ΔW and Pp decreased and increased, respectively, with increasing VPD or decreasing SWC and recovery of ΔW and Pp in the evening was impeded by low SWC. In well-watered oaks, daily positive peaking of Pp preceded daily negative peaking of ΔW; these time lags gradually increased with increasing soil drought, suggesting hydraulic uncoupling of stem and leaves.

Compartmentation of metals in foliage of Populus tremula grown on soils with mixed contamination. I. From the tree crown to leaf cell level.

In order to achieve efficient phytoextraction of heavy metals using trees, the metal allocation to aboveground tissues needs to be characterised. In his study, the distribution of heavy metals, macro- and micronutrients and the metal micro-localisation as a function of the leaf position and heavy metal treatment were analysed in poplars grown on soil with mixed metal contamination. Zinc was the most abundant contaminant in both soil and foliage and, together with cadmium, was preferentially accumulated in older foliage whereas excess copper and lead were not translocated. Changes in other element concentrations indicated an acceleration in aging as a consequence of the metal treatment. Excess zinc was irregularly accumulated inside leaf tissues, tended to saturate the veins and was more frequently stored in cell symplast than apoplast. Storage compartments including metabolically safe and sensitive subcellular sites resulted in sizable metal accumulation as well as stress reactions.

Compartmentation of metals in foliage of Populus tremula grown on soils with mixed contamination. II. Zinc binding inside leaf cell organelles.

The phytoextraction potential of plants for removing heavy metals from polluted soils is determined by their capacity to store contaminants in aboveground organs and complex them safely. In this study, the metal compartmentation, elemental composition of zinc deposits and zinc complexation within leaves from poplars grown on soil with mixed metal contamination was analysed combining several histochemical and microanalytical approaches. Zinc was the only heavy metal detected and was stored in several organelles in the form of globoid deposits showing ß-metachromasy. It was associated to oxygen anions and different cations, noteworthy phosphorous. The deposit structure, elemental composition and element ratios indicated that zinc was chelated by phytic acid ligands. Maturation processes in vacuolar vs. cytoplasmic deposits were suggested by differences in size and amounts of complexed zinc. Hence, zinc complexation by phytate contributed to metal detoxification and accumulation in foliage but could not prevent toxicity reactions therein.

Erratum to "Structural and physiological responses to ozone in Manna ash (Fraxinus ornus L.) leaves of seedlings and mature trees under controlled and ambient conditions".

Leaf-level microscopical symptom structure and physiological responses were investigated in seedlings experimentally exposed to ozone (O3) in indoor chambers (150 ppb, 8 hd(-1) per 7 weeks), and field trees of Manna ash (Fraxinus ornus) exposed to ambient O3 (max 93 ppb per one growing season). Ozone-induced leaf injury, including leaf reddening and stippling, was observed in both seedlings and mature trees, but the morphology of injury in the stipples differed, being hypersensitive-like (HR-like) in the chamber seedlings and accelerated cell senescence (ACS) in the field trees. In both exposure conditions, the main structural impact of O3 was on the mesophyll and especially the upper assimilating cell layers. The main physiological impact was on carbon assimilation and on stomatal sluggishness. These effects were not due to stomatal structural injury and were more severe in juvenile compared to mature trees because of environmental (water availability, light) and constitutional (gas exchange capacity) factors and differences in the cell physiology processes (HR-like vs. ACS) triggered by ozone stress. Given the plasticity of plant responses to ozone stress, dose/response relationships for tree seedlings in the indoor chambers cannot be extrapolated to mature trees unless ambient conditions are closely simulated.

Structural and physiological responses to ozone in Manna ash (Fraxinus ornus L.) leaves of seedlings and mature trees under controlled and ambient conditions.

Leaf-level microscopical symptom structure and physiological responses were investigated in seedlings experimentally exposed to ozone (O3) in indoor chambers (150 ppb, 8 h d(-1)/7 weeks), and field trees of Manna ash (Fraxinus ornus) exposed to ambient O3 (max 93 ppb/one growing season). Ozone-induced leaf injury, including leaf reddening and stippling, was observed in both seedlings and mature trees, but the morphology of injury in the stipples differed, being hypersensitive-like (HR-like) in the chamber seedlings and accelerated cell senescence (ACS) in the field trees. In both exposure conditions, the main structural impact of O3 was on the mesophyll and especially the upper assimilating cell layers. The main physiological impact was on carbon assimilation and on stomatal sluggishness. These effects were not due to stomatal structural injury and were more severe in juvenile compared to mature trees because of environmental (water availability, light) and constitutional (gas exchange capacity) factors and differences in the cell physiology processes (HR-like vs. ACS) triggered by ozone stress. Given the plasticity of plant responses to ozone stress, dose/response relationships for tree seedlings in the indoor chambers cannot be extrapolated to mature trees unless ambient conditions are closely simulated.

Leaf responsiveness of Populus tremula and Salix viminalis to soil contaminated with heavy metals and acidic rainwater.

Fast-growing trees such as Salix viminalis L. and Populus tremula L. are well suited to phytoremediate heavy metal contaminated soils. However, information on tree performance, particularly leaf function, under conditions of heavy metal contamination is scarce. We used yearly coppiced saplings of S. viminalis and P. tremula growing in model ecosytems to test four hypotheses: (1) heavy metal contamination impairs photosynthesis by injuring leaf structure; (2) the effects of heavy metal contamination are enhanced by acidified rainwater and low soil pH; (3) heavy metal contamination increases dark respiration and, thus, repair processes; and (4) heavy metal contamination is tolerated and remediated better by S. viminalis than by P. tremula. We investigated heavy metal accumulation, tissue injury and gas exchange in leaves of plants subjected to controlled soil contamination with heavy metal dust. Additional treatments included acidic and calcareous natural forest subsoils in combination with irrigation with rainwater at pH 5.5 or 3.5. In both provenances of P. tremula that were studied, but not in S. viminalis, heavy metal treatment reduced photosynthesis and transpiration by varying amounts, except in the hot and dry summer of 2003, but had no effect on dark respiration. At light saturation, net CO(2) uptake and water-use efficiency were reduced by heavy metal contamination, whereas the CO(2) concentration in the leaf intercellular air space was increased. Rainwater pH and subsoil pH only slightly modified the effects of the heavy metal treatment on P. tremula. Gas exchange responses of P. tremula to heavy metals were attributed to leaf structural and ultrastructural changes resulting from hypersensitive-response-like processes and accelerated mesophyll cell senescence and necroses in the lower epidermis, especially along the transport pathways of heavy metals in the leaf lamina. Overall, the effects of heavy metals on P. tremula corroborated Hypothesis 1, but refuted Hypotheses 2 and 3, and were inconclusive for Hypothesis 4. Both P. tremula and S. viminalis showed appreciable potential for storing heavy metals in aging foliage.

Linking stress with macroscopic and microscopic leaf response in trees: new diagnostic perspectives.

Visible symptoms in tree foliage can be used for stress diagnosis once validated with microscopical analyses. This paper reviews and illustrates macroscopical and microscopical markers of stress with a biotic (bacteria, fungi, insects) or abiotic (frost, drought, mineral deficiency, heavy metal pollution in the soil, acidic deposition and ozone) origin helpful for the validation of symptoms in broadleaved and conifer trees. Differentiation of changes in the leaf or needle physiology, through ageing, senescence, accelerated cell senescence, programmed cell death and oxidative stress, provides additional clues raising diagnosis efficiency, especially in combination with information about the target of the stress agent at the tree, leaf/needle, tissue, cell and ultrastructural level. Given the increasing stress in a changing environment, this review discusses how integrated diagnostic approaches lead to better causal analysis to be applied for specific monitoring of stress factors affecting forest ecosystems.

Diagnosis of abiotic and biotic stress factors using the visible symptoms in foliage.

Visible symptoms in the foliage of trees are recorded to monitor the effects of abiotic and biotic stress. Difficulties are reported in diagnosing the origin of stress. The present paper discusses several diagnostic criteria which are usable in different species for a better determination of the stress factor type. A new diagnosis scheme to differentiate between classes of abiotic and biotic stress factors is supplied. Abiotic stress generates gradients of symptoms. The symptom specificity is determined by the degree of interaction between the stress factor and plant defense system. Symptoms caused by abiotic stress and natural autumnal senescence can be morphologically different or undistinguishable according to the stress and plant species. With biotic stress, the class of parasitic is generally recognizable on the basis of the visible symptoms. Structurally and physiologically based explanations of the symptom morphology are still missing for many stress factors.