Electron donation to nitrogenase in heterocysts of cyanobacteria

Various electron donors were found to stimulate C₂H₂ reduction (N₂ fixation) by isolated heterocysts from Anabaena variabilis and Anabaena cylindrica. Intermediates of glycolysis and the tricarboxylic acid cycle as well as unphosphorylated sugars like glucose, fructose and erythrose were among these electron donors. The transfer of electrons from donors like H₂, NADH, glyoxylate and glycollate was strictly light-dependent, whereas others like NADPH or pyruvate plus coenzyme A supported C₂H₂ reduction also in the dark. In all cases, the overall activity was enhanced by light. The stimulation by light was more distinct with heterocysts from A. variabilis than with heterocysts from A. cylindrica.The present communication establishes that pyruvate supports C₂H₂ reduction by heterocysts from either A. variabilis or A. cylindrica with rates comparable to those with other electron donors. Pyruvate could, however, support C₂H₂ reduction only in the presence of coenzyme A, and the concentrations of both coenzyme A and pyruvate were crucial. A pyruvate-dependent reduction of ferredoxin by extracts from heterocysts was recorded spectrophotometrically. Glyoxylate, which is an inhibitor of thiamine pyrophosphate-dependent decarboxylations, inhibited pyruvate-dependent C₂H₂ reduction. This result supports the conclusion that pyruvate is metabolised by pyruvate: ferredoxin oxidoreductase in heterocysts. High concentrations of pyruvate and other electron donors inhibited C₂H₂ reduction which suggests that nitrogenase activity in heterocysts may be controlled by the availability of electron donors.Dedicated to Professor Norbert Pfennig, Konstanz, on the occasion of his 60th birthday     

Biochemistry and physiology of nitrogen fixation with particular emphasis on nitrogen-fixing phototrophs

Summary This paper presents an overview of aspects of N₂-fixation in phototrophic N₂-fixers. Nitrogenase is little different in phototrophs from other organisms. Evidence suggests that fixed carbon dissimilation rather than direct photoreduction from oxidised inorganic compounds or exogenous photosynthetic electron donors is the major route of reductant supply to nitrogenase in phototrophs; inRhodospirillum rubrum pyruvate is a possible electron donor to nitrogenase; in cyanobacteria the oxidative pentose phosphate pathway is important, although some recent evidence implicates glycolysis and the tricarboxylic acid cycle in reductant supply in heterocystous cyanobacteria. In photosynthetic organisms light modulation of various enzymes occurs-some Calvin cycle enzymes are light activated, some oxidative pentose phosphate pathway and glycolytic enzymes are deactivated and some tricarboxylic acid cycle enzymes are activated. Reduced levels of thioredoxin in heterocysts may contribute to the sustained functioning of the oxidative pentose phosphate pathway in heterocysts in the light and dark. In photosynthetic bacteria such asRhodospirillum rubrum an activating enzyme which removes a modifying group from inactive Fe protein can activate nitrogenase. O₂ and NH ₄ ⁺ both inhibit N₂-fixation and there is some evidence in cyanobacteria that O₂ stability of whole cell nitrogenase can be achieved by prolonged incubation of cultures at high O₂.     

Cellular compartmentation of photosynthetic and photorespiratory enzymes in the heterocystous cyanobacterium Anabaena cylindrica

Activities of enzymes of photosynthesis and photorespiration have been measured in extracts of vegetative cells and heterocysts from the filamentous cyanobacterium Anabaena cylindrica. Phosphoribulokinase, d-ribulose 1,5-bisphosphate carboxylase/oxygenase, phosphoglycollate phosphatase and glycollate dehydrogenase activities were readily measured in vegetative cell extracts, but were undetectable or negligible in heterocyst preparations. The data help to explain why heterocysts are unable to perform photosynthetic CO₂ fixation. They also exemplify the co-ordinate compartmentation of enzymes of photosynthesis and photorespiration which occur in a differentiated phototrophic prokaryote.Abbreviations Ru5P d-ribulose 5-phosphate - RuBP d-ribulose 1,5-bisphosphate - DCPIP 2,6-dichlorophenolindophenol - TES N-tris(hydroxymethyl)methyl-2-aminoethanesulphonate     

Properties of heterocysts isolated with colloidal silica

A method is described for the isolation of heterocysts that are virtually free of contaminating cell debris after sonication of aerobically grown Anabaena 7120. Isolated heterocysts reduced acetylene in a light-dependent process in the absence of exogenously provided ATP; heterocysts supplied with ATP and Na₂S₂O₄ reduced acetylene slowly in the dark but still showed a marked light activation. Nitrogenase activity was greatest in fractions containing intact heterocysts. Up to 13% of the activity of the intact filaments was accounted for in the isolated heterocyst preparation.Isolated heterocysts took up O₂ in a light-independent process; O₂ uptake with added NADP⁺ was enhanced by pyruvate, isocitrate and intermediates of the oxidative pentose pathway.     

Dynamics and Cell-Type Specificity of the DNA Double-Strand Break Repair Protein RecN in the Developmental Cyanobacterium Anabaena sp. Strain PCC 7120

DNA replication and repair are two fundamental processes required in life proliferation and cellular defense and some common proteins are involved in both processes. The filamentous cyanobacterium Anabaena sp. strain PCC 7120 is capable of forming heterocysts for N₂ fixation in the absence of a combined-nitrogen source. This developmental process is intimately linked to cell cycle control. In this study, we investigated the localization of the DNA double-strand break repair protein RecN during key cellular events, such as chromosome damaging, cell division, and heterocyst differentiation. Treatment by a drug causing DNA double-strand breaks (DSBs) induced reorganization of the RecN focus preferentially towards the mid-cell position. RecN-GFP was absent in most mature heterocysts. Furthermore, our results showed that HetR, a central player in heterocyst development, was involved in the proper positioning and distribution of RecN-GFP. These results showed the dynamics of RecN in DSB repair and suggested a differential regulation of DNA DSB repair in vegetative cell and heterocysts. The absence of RecN in mature heterocysts is compatible with the terminal nature of these cells.     

Observation of microplasmodesmata in both heterocyst-forming and non-heterocyst forming filamentous cyanobacteria by freeze-fracture electron microscopy

Structures which may establish cytoplasmic continuity between adjacent cells of filamentous cyanobacteria have been observed by freeze-fracture electron microscopy. They are visible in the septum region of the plasma membrane as pits on the E-face (EF) and corresponding protrusions on the P-face (PF). Between 100 and 250 of these structures, termed microplasmodesmata, were present between adjacent vegetative cells in all four strains of heterocyst-forming filamentous cyanobacteria, Anabaena cylindrica Lemm, A. variabilis (IUCC B377), A. variabilis Kütz. (ATCC 29413) and Nostoc muscorum, examined. Only 30–40 microplasmodesmata were observed between adjacent cells in two species, Phormidium luridum and Plectonema boryanum, that do not form heterocysts. The results suggest that in species that form heterocysts a greater degree of cytoplasmic continuity is established, presumably to facilitate the exchange of metabolites. In species capable of forming heterocysts, the number of microplasmodesmata per septum between two adjacent vegetative cells remained constant whether the filaments were grown in the presence of NH₄ and lacked heteroxysts or under N₂-fixing conditions and contained heterocysts. When a vegetative cell differentiates into a heterocyst, about 80% of the existing microplasmodesmata are destroyed as the poles of the cell become constricted into narrow necks leaving smaller areas of contact with the adjacent vegetative cells.     

Hydrogen metabolism in isolated heterocysts of Anabaena 7120

Isolated heterocysts of Anabaena 7120 evolve H₂ in an ATP-dependent nitrogenase-catalyzed process that is inhibited by N₂ and C₂H₂. Heterocysts have an active uptake hydrogenase that only requires an electron acceptor of positive redox potential, e.g., methylene blue, dichlorophenolindophenol or potassium ferricyanide. O₂ supplied at low partial pressures is a very effective physiological oxidant for H₂ uptake. High concentrations of O₂ are inhibitory to H₂ uptake. The oxyhydrogen reaction in heterocysts appears to be mediated by a cytochrome-cytochrome oxidase system, and it supports ATP synthesis via oxidative phosphorylation. Attempts to demonstrate acetylene reduction in isolated heterocysts employing H₂ as an electron donor were unsuccessful. It is suggested that the uptake hydrogenase functions to conserve reductant that otherwise would be dissipated via nitrogenase-catalyzed H₂ evolution.     

Pigment distribution and nitrogen fixation inAnabaena azollae

Summary The symbiotic heterocystous cyanobacteriumAnabaena azollae present in the leaf cavities of the water fernAzolla spp. was studied. The cyanobacteria extracted from the leaf cavities showed differences in pigment composition in three species ofAzolla, i.e A.pinnata var.pinnata, A.caroliniana and A.filiculoides, as observed by pigment absorption and epifluorescence tests. These differences suggest that of these species the cyanobiont ofA. pinnata is the most actively nitrogenfixing form. This has been confirmed by nitrogen fixation (acetylene reduction) tests. Heterocysts of the symbiont ofA. pinnata were characterized by high chlorophylla and low phycocyanin content, a low fluorescence yield of chlorophyll in the heterocysts compared to vegetative cells and a gradient of phycocyanin concentration in the vegetative cells adjacent to heterocysts. This indicates that only photosystem I is present in the heterocyst. In the two otherAzolla species quantitative shifts in the pigment composition occurred suggesting a lower nitrogen fixation activity.In the cyanobiontAnabaena azollae the heterocyst frequency could reach a value of 44–45%. It is argued that there are two generations of heterocysts in a matureAzolla plant, which are concomitant with two peaks of nitrogen fixation activity correlated with leaf age,i.e. leaf number along the main axis of the plant. At both peaks of maximal N₂-ase activity, only 20–25% of the heterocysts present are metabolically active as demonstrated by the reduction of Neotetrazolium chloride (NTC) in the heterocysts and darkening of nuclear emulsions by silver salt reduction. Vegetative cells of the cyanobiont reduce Neotetrazolium chloride (NTC) to formazan more rapidly than has been observed in the free-living heterocystous cyanobacteriumAnabaena cylindrica tested in parallel experiments. This feature may be due to a more permeable cell wall of the vegetative cells of the cyanobiont compared to the free-living form, since the vegetative cells of the symbiont play a role in cross-feeding of the host (Azolla).Evidence is obtained that only the heterocysts of the cyanobiont ofAzolla are involved in the nitrogen fixation process as in free-living heterocystous cyanobacterium species. This situation is different from other cyanobacterial symbioses such as inGunnera, Blasia andAnthoceros, where physiological modifications are reported in the symbiosis with another photosynthetic partner such as the absence of O₂ evolution and the absence of photo-fixation of CO₂ in the cyanobionts.Pigment composition and N₂-ase activity in the symbiotic cyanobacteria of three Azolla species have indicated the superiority of theA. pinnata symbiont.A. pinnata var.pinnata is a semidomesticated form used in S.E. Asia for agricultural purposes (irrigated rice culture) to increase soil fertility.It is suggested that by selection (domestication) more efficient strains (clones) can be obtained, and further that with more advanced techniques such as gene mutation and genetic manipulation even more efficient and for agriculture more beneficial clones can be obtained.     

Isolation of cyanobacterial heterocysts with high and sustained dinitrogen-fixation capacity supported by endogenous reductants

A method is described for the preparation of cyanobacterial heterocysts with high nitrogen-fixation (acetylene-reduction) activity supported by endogenous reductants. The starting material was Anabaena variabilis ATCC 29413 grown in the light in the presence of fructose. Heterocysts produced from such cyanobacteria were more active than those from photoautotrophically-grown A. variabilis, presumably because higher reserves of carbohydrate were stored within the heterocysts. It proved important to avoid subjecting the cyanobacteria to low temperatures under aerobic conditions, as inhibition of respiration appeared to lead to inactivation of nitrogenase. Low temperatures were not harmful in the absence of O₂. A number of potential osmoregulators at various concentrations were tested for use in heterocyst isolation. The optimal concentration (0.2M sucrose) proved to be a compromise between adequate osmotic protection for isolated heterocysts and avoidance of inhibition of nitrogenase by high osmotic strength. Isolated heterocysts without added reductants such as H₂ had about half the nitrogen-fixation activity expected on the basis of intact filaments. H₂ did not increase the rate of acetylene reduction, suggesting that the supply of reductant from heterocyst metabolism did not limit nitrogen fixation under these conditions. Such heterocysts had linear rates of acetylene reduction for at least 2 h, and retained their full potential for at least 12 h when stored at 0°C under N₂.     

Effect of ammonia and sulfide on rifampicin-induced heterocyst formation inNostoc linckia

The effect of ammonia and sulfide on rifampicin-induced heterocyst differentiation was studied in the nitrogen-fixing cyanobacteriumNostoc linckia. Aerobic growth with nitrogen gas of the cyanobacterium was greatly affected by rifampicin with formation of multiple heterocysts in chains in the filaments whereas ammonia in the medium reversed the rifampicin inhibition of growth and prevented the induction of heterocysts. In a sulfide medium the suppression exerted by rifampicin on aerobic growth with nitrogen gas and heterocyst induction was found to be considerably reduced. The results suggest two interesting points,viz. that (i) rifampicin interferes with the nitrogen-fixing function of heterocysts, and (ii) it checks the synthesis of an unknown heterocyst, inhibitor and thus permits the adjacent vegetative cells to differentiate into heterocysts in chains.     

Modes of electron transfer from molecular hydrogen in Anabaena cylindrica

Several natural and artificial electron donors were assayed in the C₂H₂-reduction of heterocysts isolated from the cyanobacterium Anabaena cylindrica. Among these, molecular hydrogen was the most effective one when the assays were performed in the light. The C₂H₂-reduction and the Knallgas reaction of intact Anabaena filaments as well as the H₂-supported C₂H₂-reduction of isolated heterocysts were compared for their sensitivity towards several inhibitors known to affect the photosynthetic or respiratory electron flow. Among these, dibromothymoquinone (DBMIB) affected all three reactions equally indicating that plastoquinone is a common intermediate of the H₂-consumptions by either the respiratory or the photosynthetic electron transport. Metronidazole inhibited the H₂-utilization via photosynthesis but did not affect the consumption of this gas by respiration and therefore allows to differentiate between the two pathways of hydrogen utilization. The studies with the inhibitors are suggestive for a segment of electron carriers on the membranes common to both photosynthesis and respiration in heterocysts of Anabaena.Abbreviations BNT 2-bromo-4-nitrothymol - DAD diaminodurene - DBMIB 2,5-dibromothymoquinome - DCMU dichlorophenyl dimethylurea - DCPIP dichlorophenol indophenol - DMSO dimethylsulphoxide - TMPD N-tetramethyl-p-phenylenediamine - chl chlorophyll     

Localized Tetrazolium Reduction in Relation to N2 Fixation, CO2 Fixation, and H2 Uptake in Aquatic Filamentous Cyanobacteria

The aquatic filamentous cyanobacteria Anabaena oscillarioides and Trichodesmium sp. reveal specific cellular regions of tetrazolium salt reduction. The effects of localized reduction of five tetrazolium salts on N₂ fixation (acetylene reduction), ¹⁴CO₂ fixation, and ³H₂ utilization were examined. During short-term (within 30 min) exposures in A. oscillarioides, salt reduction in heterocysts occurred simultaneously with inhibition of acetylene reduction. Conversely, when salts failed to either penetrate or be reduced in heterocysts, no inhibition of acetylene reduction occurred. When salts were rapidly reduced in vegetative cells, ¹⁴CO₂ fixation and ³H₂ utilization rates decreased, whereas salts exclusively reduced in heterocysts were not linked to blockage of these processes. In the nonheterocystous genus Trichodesmium, the deposition of reduced 2,3,5-triphenyl-2-tetrazolium chloride (TTC) in the internal cores of trichomes occurs simultaneously with a lowering of acetylene reduction rates. Since TTC deposition in heterocysts of A. oscillarioides occurs contemporaneously with inhibition of acetylene reduction, we conclude that the cellular reduction of this salt is of use in locating potential N₂-fixing sites in cyanobacteria. The possible applications and problems associated with interpreting localized reduction of tetrazolium salts in cyanobacteria are presented.     

Cellular localization of cytochrome c 553 in the N2-fixing cyanobacterium Anabaena variabilis

The in situ location of the electron carrier protein cytochrome C ₅₅₃ (cyt c ₅₅₃) has been investigated in both vegetative cells and heterocysts of the cyanobacterium Anabaena variabilis ATCC 29413 using the antibody-gold technique, carried out as a post-ernbedding immunoelectron microscopy procedure. When using a rabbit polyclonal anti-cyt c ₅₅₃ specific antiserum an intense labelling, associated mainly with the cell periphery (cytoplasmic membrane and periplasmic area), was seen in both heterocysts and vegetative cells. The selective release of most of the cellular cyt c ₅₅₃ during a Tris-EDTA treatment confirms a periplasmic localization of this protein in A. variabilis. The results indicate that most of cyt c ₅₅₃ is located in the periplasmic space. The roles ascribed to this protein in both respiration and photosynthesis in cyanobacteria are discussed.Abbreviations Cyt c ₅₅₃ cytochrome c ₅₅₃ - PBS phosphate buffered saline (20 mM sodium phosphate, 0.9% NaCl, pH 7.4) - PMSF phenylmethylsulfonyl fluoride Recipient of a Research Fellowship of the Alexander von Humboldt Foundation (Bonn, FRG) for a leave to the University of Konstanz.     

Correlation between nitrogenase and glutamine synthetase expression in the cyanobacterium Anabaena cylindrical

Distribution pattern and levels of nitrogenase (EC 1.7.99.2) and glutamine synthetase (GS, EC 6.3.1.2) were studied in N₂-, NO₃^? and NH₄⁺ grown Anabaena cylindrica (CCAP 1403/2a) using immunogold electron microscopy. In N₂- and NO₃^? grown cultures, heterocysts were formed and nitrogenase activity was present. The nitrogenase antigen appeared within the heterocysts only and showed an even distribution. The level of nitrogenase protein in the heterocysts was identical with both nitrogen sources. In NO₃^? grown cells the 30% reduction in the nitrogenase activity was due to a corresponding decrease in the heterocyst frequency and not to a repressed nitrogenase synthesis. In NH₄^? grown cells, the nitrogenase activity was almost zero and new heterocysts were formed to a very low extent. The heterocysts found showed practically no nitrogenase protein throughout the cytoplasm, although some label occurred at the periphery of the heterocyst. This demonstrates that heterocyst differentiation and nitrogenase expression are not necessarily correlated and that while NH₄⁺ caused repression of both heterocyst and nitrogenase synthesis, NO₃^? caused inhibition of heterocyst differentiation only. The glutamine synthetase protein label was found throughout the vegetative cells and the heterocysts of all three cultures. The relative level of the GS antigen varied in the heterocysts depending on the nitrogen source, whereas the GS level was similar in all vegetative cells. In N₂- and NO₃⁺ grown cells, where nitrogenase was expressed, the GS level was ca 100% higher in the heterocysts compared to vegetative cells. In NH₄⁺ grown cells, where nitrogenase was repressed, the GS level was similar in the two cell types. The enhanced level of GS expressed in heterocysts of N₂ and NO₃^? grown cultures apparently is related to nitrogenase expression and has a role in assimilation of N₂derived ammonia.     

Overexpression of SepJ alters septal morphology and heterocyst pattern regulated by diffusible signals in Anabaena

Filamentous, N₂‐fixing, heterocyst‐forming cyanobacteria grow as chains of cells that are connected by septal junctions. In the model organism Anabaena sp. strain PCC 7120, the septal protein SepJ is required for filament integrity, normal intercellular molecular exchange, heterocyst differentiation, and diazotrophic growth. An Anabaena strain overexpressing SepJ made wider septa between vegetative cells than the wild type, which correlated with a more spread location of SepJ in the septa as observed with a SepJ–GFP fusion, and contained an increased number of nanopores, the septal peptidoglycan perforations that likely accommodate septal junctions. The septa between heterocysts and vegetative cells, which are narrow in wild‐type Anabaena, were notably enlarged in the SepJ‐overexpressing mutant. Intercellular molecular exchange tested with fluorescent tracers was increased for the SepJ‐overexpressing strain specifically in the case of calcein transfer between vegetative cells and heterocysts. These results support an association between calcein transfer, SepJ‐related septal junctions, and septal peptidoglycan nanopores. Under nitrogen deprivation, the SepJ‐overexpressing strain produced an increased number of contiguous heterocysts but a decreased percentage of total heterocysts. These effects were lost or altered in patS and hetN mutant backgrounds, supporting a role of SepJ in the intercellular transfer of regulatory signals for heterocyst differentiation.     

The relation of heterocysts and hormogonia to nitrogen fixation and reproduction in blue-green algae

Indications are presented that the heterocysts of a member of theRivulariaceae germinate in NO₃ free medium containing low concentrations of NH₄Cl and glucose. Growth occurred regularly in the absence of combined nitrogen sources (NH₄, NO₃), suggesting N₂ fixation. Phosphate regulates hormogonia release, perhaps indirectly via an influence on differentiation of heterocysts.     

Proteome Analysis of Enriched Heterocysts from Two Hydrogenase Mutants from Anabaena sp. PCC 7120

Cyanobacteria are oxygenic photosynthetic prokaryotes and play a crucial role in the Earth's carbon and nitrogen cycles. The photoautotrophic cyanobacterium Anabaena sp. PCC 7120 has the ability to fix atmospheric nitrogen in heterocysts and produce hydrogen as a byproduct through a nitrogenase. In order to improve hydrogen production, mutants from Anabaena sp. PCC 7120 are constructed by inactivation of the uptake hydrogenase (ΔhupL) and the bidirectional hydrogenase (ΔhoxH) in previous studies. Here the proteomic differences of enriched heterocysts between these mutants cultured in N₂‐fixing conditions are investigated. Using a label‐free quantitative proteomics approach, a total of 2728 proteins are identified and it is found that 79 proteins are differentially expressed in the ΔhupL and 117 proteins in the ΔhoxH variant. The results provide for the first time comprehensive information on proteome regulation of the uptake hydrogenase and the bidirectional hydrogenase, as well as systematic data on the hydrogen related metabolism in Anabaena sp. PCC 7120.     

Tetrazolium reduction and nitrogenase activity in heterocystous blue-green algae

Summary Heterocysts reduce triphenyl tetrazolium chloride (TTC) faster than vegetative cells apparently because the absence of the O₂-evolving photosystem II and the high electron transport activity in these cells. Although the rate of TTC reduction in vegetative cells is increased by the continuous removal of O₂ evolved in photosynthesis, it has not been possible to obtain rates of TTC reduction comparable with those in heterocysts probably because of the continued competition for electrons between TTC and O₂. The use of nitro-blue tetrazolium chloride (NBT) as a redox indicator has revealed the presence in filaments under aerobic conditions of a gradient of electron transport activity with strongest reducing power in the heterocysts, proheterocysts and vegetative cells next to heterocysts, and with gradually diminishing activity midway between two heterocysts. This pattern is indistinct in filaments grown under micro-aerophilic conditions. The strong electron transport activity in vegetative cells adjacent to heterocysts appears to promote reducing conditions in the heterocysts. Both, red-formazan formation in the heterocysts and blue-formazan deposition in vegetative cells greatly inhibit nitrogenase activity, and this was adversely affected also by the detachment of heterocysts from vegetative cells. The findings are consistent with the idea that the association of heterocysts with vegetative cells in essential for nitrogen fixation to occur in heterocystous blue-green algae.     

MEK/ERK inhibitor U0126 enhanced salt stress-induced programmed cell death in <Emphasis Type="Italic">Thellungiella halophila</Emphasis> suspension-cultured cells

Programmed cell death (PCD) is an active cellular suicide that occurs both in animals and plants throughout development and in response to abiotic or biotic stress. In contrast to plant hypersensitive response-like cell death, little is known about the molecular machinery that regulates the halophyte plant PCD under high salinity stress. Since mitogen-activated protein kinases (MAPKs) are involved in plant response/tolerance to salt stress, and plant MAPK genes belong to the extracellular signal-regulated kinase (ERK) subfamily, we have investigated the role of ERK-like enzymes in high salinity stress-induced cell death in Thellungiella halophila. The data showed that ERK-like enzymes were early (10 min) and transiently activated under 300 mM NaCl stress. Pretreatment with 10 μM U0126, a special MEK/ERK inhibitor, resulted in a small but statistically significant increase of the percentage of terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling (TUNEL)-positive nuclei in contrast to salt alone. The effects of U0126 on H₂O₂ production and cytochrome c (cyt c) release were also investigated. We found that the pretreatment with U0126 accelerated H₂O₂ production as well as cyt c release, and eventually enhanced cell death. The results suggest that ERK-like enzymes in Thellungiella halophila may act as a positive regulator of salt tolerance, as illustrated by pretreatment with U0126 which enhanced cell death under high salinity stress.