Carbonic Anhydrase,Photosynthesis, and Seed Yield in Mustard Plants Treated with Phytohormones

The leaves of 30-d-old plants of Brassica juncea Czern & Coss cv. Varuna were sprayed with 10^–6 M aqueous solutions of indole-3-yl-acetic acid (IAA), gibberellic acid (GA₃), kinetin (KIN), and abscisic acid (ABA) or 10^–8 M of 28-homobrassinolide (HBR). All the phytohormones, except ABA, improved the vegetative growth and seed yield at harvest, compared with those sprayed with deionised water (control). HBR was most prominent in its effect, generating 32, 30, 36, 70, 25, and 29 % higher values for dry mass, chlorophyll content, carbonic anhydrase (E.C. 4.2.1.1) activity, and net photosynthetic rate in 60-d-old plants, pods per plant, and seed yield at harvest, over the control, respectively. The order of response to various hormones was HBR > GA₃ > IAA > KIN > control > ABA.     

Salicylic Acid Influences Net Photosynthetic Rate,Carboxylation Efficiency,Nitrate Reductase Activity,and Seed Yield in Brassica juncea

Aqueous solutions of salicylic acid (SA) were applied to the foliage of 30-d-old plants of mustard (Brassica juncea Czern & Coss cv. Varuna). The plants sprayed with the lowest used concentration (10₋₅ M) of SA were healthier than those sprayed with water only or with higher concentrations of SA (10⁻⁴ or 10⁻³ M). 60-d-old plants possessed 8.4, 9.8, 9.3, 13.0 and 18.5 % larger dry mass, net photosynthetic rate, carboxylation efficiency, and activities of nitrate reductase and carbonic anhydrase over the control, respectively. Moreover, the number of pods and the seed yield increased by 13.7 and 8.4 % over the control. This revised version was published online in August 2006 with corrections to the Cover Date.     

Photosynthetic Response of Vigna radiata to Pre-Sowing Seed Treatment with 28-Homobrassinolide

Surface sterilised seeds of mungbean (Vigna radiata L. Wilczek cv. T-44) were soaked in 0, 10⁻⁸, 10⁻⁶, or 10⁻⁴ M aqueous solution of 28-homobrassinolide (HBR) for 4, 8, or 12 h. The treated seeds were grown in sandy loam soil filled in earthen pots and sampled at 30, 40, and 50 d. Net photosynthetic rate, leaf chlorophyll content, carbonic anhydrase activity (E.C. 4.2.1.1), carboxylation efficiency, stomatal conductance, and seed yield at harvest were enhanced by the HBR treatment. The best combination was the pre-sowing seed treatment with 10⁻⁶ M HBR for 8 h. This revised version was published online in August 2006 with corrections to the Cover Date.     

Trade-off relationships between some reproductive characteristics in plants with special reference to life history stragegy

Several reproductive triats in plants were studied in more than 200 populations of 61 wild species from diverse ecological conditions. As a result, it was found that there occur three distinct types of plants in the energy allocation patterns to reproductive structures (RA) and the propagule output per plant (P_N), i.e. (1) the number of propagules per plant increases in response to the increase in RA (Type I), (2) the number of propagules decreases in response to the increase in RA (Type II), and (3) the RA remains constant despite the great differences in the propagule number per plant. A conspicuous trade-off relationship was also discovered to occur between the RA to a single propagule (R_A) and the propagule output per plant (P_N), such that log R_A=logC−blot P_N, or R_A=C/P_N ^b =CP_N ^−b , where C is a constant. The three different ranges ofb-values were recognized, i.e.b<1.0,b>1.0, andb=1.0, which correspond to Type I, Type II, and Type III, respectively. Related problems to the concept ofr- andK-strategy are also discussed.     

Callus induction and plantlet regeneration in Bixa oreliana L., an annatto-yielding tree

Summary A protocol has been developed for plantlet regeneration from seed callus of Bixa orellana L. Seeds demonstrated a high percentage of callus induction (63±7.3%) and a high yield (356±14.7 mg per seed) of white friable callus on Murashige and Skoog (MS) medium containing 5.0 μM l-naphthaleneacetic acid (NAA) and 2.5μM N ⁶-benzyladenine (BA) within 6 wk of culture in the dark. Callus induction frequency was greater under 24h dark as compared to 16h light/8h dark photoperiod or 24h light photoperiod. Increased myo-inositol (MI: 200mgl⁻¹) and addition of ascorbic acid (AA: 200 mgl⁻¹) to the culture medium positively improved callus induction frequency and growth. Shoot differentiation from white friable seed callus was best using 10.0 μM BA and 5.0 μM NAA, where the highest percentage of calluses forming shools (74.9±4.8%), the highest number of shoots per callus (six or seven) and the highest shoot-forming index (5.0) were obtained within 6 wk. Shoots elongated to 4 cm within 4 wk of transfer onto MS medium devoid of growth regulators. Shoots were rooted using half-strength MS medium containing 5.0 μM indole-3-butyric acid (IBA). About 85% of these plants were established in pots containing pure garden soil and organic manure after 3 wk of hardening. Regenerated plants were morphologically uniform with normal leaf, shape and growth patterns. These plants are currently being screened for the presence of agronomically useful genetic variants.     

Nitrogen fixation and growth of soybean as influenced by varying the methods of inoculation with Bradyrhizobium japonicum

The effects of inoculating soil with a water suspension of Bradyrhizobium japonicum (i) at seeding, (ii) 7, or (iii) 14 days after planting (DAP), (iv) seed slurry inoculation and (v) seed slurry supplemented with postemergence inoculation of a water suspension of Bradyrhizobium at 7 or (vi) 14 DAP, on nodulation, N₂ fixation and yield of soybean (Glycine max. [L.] Merrill) were compared in the greenhouse. The ¹⁵N isotope dilution technique was used to quantify N₂ fixed at flowering, early pod filling and physiological maturity stages (36, 52 and 70 DAP, respectively). On average, the water suspension inoculation formed the greatest number of nodules, and seed plus postemergence inoculation formed slightly more nodules than the seed-only inoculated plants (27, 19 and 12 nodules/plant respectively at physiological maturity). Seed slurry inoculation followed by postemergence inoculation at 14 DAP gave the highest nodule weight, with the plants fixing significantly more (P<0.05) N₂ (125 mg N plant⁻¹ or 56% N) than any other treatment (mean, 75 mg plant⁻¹ or 35% N). However, the higher N₂ fixation was not translated into higher N or dry matter yields. Estimates of N₂ fixed by the ostemergence Bradyrhizobium inoculations as well as plant yield were not significantly different from those of the seed slurry inoculation. Thus, delaying inoculation (e.g., by two weeks as in this study) did not reduce the symbiotic ability of soybean plants.     

Effects of zinc and soil moisture on photosynthetic rate and chlorophyll fluorescence parameters of maize

Effects of zinc [0 and 5.0 mg Zn kg⁻¹ (soil)] on photosynthetic rate (P_N), and chlorophyll fluorescence in leaves of maize (Zea mays L.) cv. Zhongdan 9409 seedlings grown under different soil moisture regimes (40–45 % and 70–75 % of soil saturated water content) were studied. Zn application did not enhance maize plant adaptation to drought stress. The relative water content and the water potential of leaves were not affected by Zn treatment. Moreover, The P_N of drought-stressed plants was not improved by Zn supply. The increases of plant biomass, stomatal conductance and quantum yield of photosystem 2 due to Zn addition were notable in well-watered plants.     

Growth and photosynthetic characteristics of <Emphasis Type="Italic">Nigella sativa</Emphasis> L. as affected by presowing seed treatment with kinetin

Black cumin (Nigella sativa L.) seeds were surface-sterilized and soaked for 5, 10, or 15 h in 1, 10, or 100 μM aqueous solution of kinetin (KIN). The potted plants were then analyzed at 30, 50, and 70 days after emergence (DAE) for dry mass (DM), leaf area (LA), chlorophyll (Chl) content, stomatal conductance (g _s), carbonic anhydrase (CA), and nitrate reductase (NR) activity, total protein content, and net photosynthetic rate (P _N). Capsule number and seed yield were determined at harvest (90 DAE). Treatment with the growth regulator was found to appreciably enhance all the determinants, with most prominent results being obtained following 10-h soaking with 10 μM KIN, in which case the values for DM, LA, P _N, CA and NR activity, and seed yield were elevated by 55, 63, 43, 38, 29, and 23% respectively over the control at the 50-day stage.     

Contribution of plants to N 2 O emissions in soil-winter wheat ecosystem: pot and field experiments

Outdoor pot and field experiments were conducted to assess the role of growing plants in agricultural ecosystem N₂O emissions. N₂O emissions from plants were quantified as the difference in soil-crop system N₂O emissions before and immediately after cutting plants during the main growth stages in 2001–02 and 2002–03 winter wheat seasons. Emissions of N₂O from plants depended on biomass within the same plant developmental status. Field results indicated that the seasonal contribution of N₂O emissions from plants to ecosystem fluxes averaged 25%, ranging from 10% at wheat tillering to 62% at the heading stage. The fluxes of N₂O emissions from plants varied between 0.3 and 3.9 mg N₂O-N m⁻² day⁻¹ and its seasonal amount was equivalent to 0.23% of plant N released as N₂O. A N₂O emission coefficient (N₂O_E, mg N₂O-N g⁻¹ C day⁻¹), defined as N₂O-N emission in milligrams from per gram carbon of plant dry matter within a day, was represented by a 5-fold variation ranging from 0.021 to 0.004 mg N₂O-N g C⁻¹ day⁻¹. A linear relationship (y=0.4611x+0.0015, r ²=0.9352, p < 0.001) between N₂O_E (y) and plant dark respiration rate (x, mg CO₂-C g C⁻¹ day⁻¹) suggested that in the absence of photosynthesis, some N₂O production in plant N assimilation was associated with plant respiration. Although this study could not show whether N₂O was produced or transferred by winter wheat plants, these results indicated an important role for higher plant in N₂O exchange. Identifying its potential contribution is critical for understanding agricultural ecosystem N₂O sources.     

Aerial seed storage in <Emphasis Type="Italic">Melaleuca ericifolia</Emphasis> Sm. (Swamp Paperbark): environmental triggers for seed release

Aerial seed banks are potentially the main source of sexual recruitment for woody wetland plants. Whilst the importance of soil seed banks for the persistence and recruitment of wetland plants has been examined in many studies, the role of aerial seed banks has been largely neglected. We used seed traps and the seedling emergence technique to quantify the seed rain from aerial seed banks of the Swamp Paperbark Melaleuca ericifolia Sm. (Myrtaceae) in Dowd Morass, a Ramsar-listed, brackish-water wetland in south-eastern Australia. Nine plant species germinated from material collected in seed traps over 2004–2005, but emergents were dominated (80–97%) by M. ericifolia. The mean number of M. ericifolia emergents ranged from <1 to <100 seedlings m⁻² day⁻¹, and showed a peak in the summer–autumn period. Regression analysis showed a significant negative correlation (r ² = 0.738) between the number of M. ericifolia emergents and water depth. Water depth and salinity were negatively correlated (r ² = 0.819), and increases in the number of M. ericifolia emergents as water levels fell were also associated with high salinities. Increasing air temperature and vapour pressure deficit also stimulated seed release during periods of drying. This study is one of the first to demonstrate the importance of aerial seed banks for sexual recruitment in woody wetland plants and the release of seed in relation to environmental factors. Aerial seed banks warrant consideration alongside soil seed banks for the establishment and long-term survival of woody plants in wetlands. Handling editor: Luis Mauricio Bini     

Responses of <Emphasis Type="Italic">Nigella sativa</Emphasis> to foliar application of gibberellic acid and kinetin

Foliar sprays of water or 1, 10 and 100 μM aqueous solutions of gibberellic acid (GA₃) or kinetin (KIN) were applied to 40-d-old plants of Nigella sativa (L.) to study their effects on net photosynthetic rate, nitrogen metabolism, and the seed yield. 10 μM solutions of both the hormones, especially GA₃, appreciably increased the activities of nitrate reductase and carbonic anhydrase, chlorophyll and total protein contents and net photosynthetic rate in the leaves, along with capsule number and seed yield plant⁻¹, at harvest.     

Photosynthesis and dark respiration of leaves of terrestrial carnivorous plants

Using CO₂ gasometry, net photosynthetic (P _N) and dark respiration rates (R _D) were measured in leaves or traps of 12 terrestrial carnivorous plant species usually grown in the shade. Generally, mean maximum P _N (60 nmol CO₂ g⁻¹(DM) s⁻¹ or 2.7 μmol m⁻² s⁻¹) was low in comparison with that of vascular non-carnivorous plants but was slightly higher than that reported elsewhere for carnivorous plants. After light saturation, the facultatively heliophytic plants behaved as shade-adapted plants. Mean R _D in leaves and traps of all species reached about 50% of maximum P _N and represents the high photosynthetic (metabolic) cost of carnivory.     

The relative contribution of symbiotic N2 fixation and other nitrogen sources to grassland ecosystems along an altitudinal gradient in the Alps

The significance of symbiotic N₂ fixation in legumes (Trifolium alpinum L., T. nivale Sieber, T. pratense L., T. badium Schreber, T. thalii Vill., T. repens L., Lotus alpinus [DC.] Schleicher, L. corniculatus L., Vicia sativa L.) and other N sources for the N budget of grassland ecosystems was studied along an altitudinal gradient in the Swiss Alps. The total annual symbiotic N₂ fixation was compared with other sources of N for plant growth of the total plant community (mineralisation and wet deposition). The contribution of symbiotically fixed N to total above-ground N yield of the swards decreased from at least 16% to 9% with increasing altitude where legumes were present. This decrease was due to a decrease in the yield proportion of legumes from 15% at 900 and 1380 m a.s.l. to 5% at 2100 and 2300 m a.s.l. (no legumes were found above 2750 m a.s.l.) and not to a decline in the activity of symbiotic N₂ fixation. With increasing altitude legumes are more patchily distributed. The high symbiotic N₂ fixation of individual plants up to their altitudinal limit is not primarily the result of low mineral N availability since an addition of NH₄ ⁺ or NO₃ ⁻ fertiliser at 2300 m a.s.l. led either to no decrease or only to a minor decrease in symbiotic N₂ fixation. At 1380 m a.s.l., N mineralisation (13.45 g N m⁻² yr⁻¹) appeared to be the main source of N for growth of the sward; N from symbiosis (at least 1.0 g to 2.6 g N m⁻² yr⁻¹) and wet deposition (0.4 g to 0.6 g m⁻² yr⁻¹) was not a significant N source for plant growth at this altitude. At 2100 m a.s.l., the combined amounts of N from symbiotic N₂ fixation (at least 0.1 g N m⁻² yr⁻¹) and wet deposition (0.3 g N m⁻² yr⁻¹) appeared to be similarly important for plant growth as soil N mineralisation (0.47 g N m⁻² yr⁻¹). At high altitudes, wet N deposition and symbiotic N₂ fixation together represent a significant source of N for the grassland ecosystem while at low altitudes these N inputs appear to be much less important. This revised version was published online in June 2006 with corrections to the Cover Date.     

Effect of gibberellic acid on carbonic anhydrase,photosynthesis, growth and yield of mustard

The plants of mustard (Brassica juncea L.) were treated with 0, 25 and 50 ΜM gibberellic acid (GA₃) at three fully developed leaf stage (30 d after sowing). Effect of GA₃ on carbonic anhydrase activity, photosynthetic rate, leaf area index and dry mass was studied at 50, 70 and 90 d after sowing. At harvest 1000 seed mass, pod number and seed yield were recorded. GA₃ treatment (50 ΜM) enhanced all the characteristics studied.     

Nitrogen use efficiency of sugarcane in relation to its BNF potential and population of endophytic diazotrophs at different N levels

The nitrogen fixing bacterial endophytes Gluconacetobacter diazotrophicus and Herbaspirillum spp. have been proposed to benefit sugarcane (Saccaharum spp. hybrids) growth. Variable populations of these endophytes exist depending upon ontogenic and climatic variations as well. This study investigates the effect of variable chemical nitrogen application in soil on the population of endophytic diazotrophs, acetylene reduction ability of excised roots, plant N-nutrient use efficiency and probable interactions among different parameters in eight commercial sugarcane varieties of subtropical India. Recovery efficiency (RE), agronomic efficiency (AE), partial factor productivity (PFP) and physiologic efficiency (PE) indicators were used for accounting N-nutrient use efficiency. The population of G. diazotrophicus was more at N₇₅ compared to N₀ and N₁₅₀, whereas Herbaspirillum population increased from N₀ to N₁₅₀. ARA was positively correlated with Gluconacetobacter population in rhizosphere and root, whereas it had poor correlation with Herbaspirillum population. Positive correlation of RE and AE with ARA of roots, Gluconacetobacter and Herbaspirillum populations in roots and stems indicate their positive contribution in total nitrogen uptake by the plant per kg of N applied. Average PFP was 808.9 at N₇₅ compared to 408.7 at N₁₅₀ indicating that N was utilized efficiently at low N input status in sugarcane. Strong positive correlations of AE₇₅ (agronomic efficiency from 75 kg N ha⁻¹ to 150 kg N ha⁻¹) with N-uptake (r ² = 0.615), cane yield (r ² = 0.758) and PFP (r ² = 0.758) and other parameters compared to AE (agronomic efficiency from 0 kg N ha⁻¹ to 75 kg N ha⁻¹ or 150 kg N ha⁻¹) correlations with N-uptake (r ² = 0.111), cane yield (r ² = 0.368) and PFP (r ² = 0.190) indicated that the AE of sugarcane was strongly directed towards producing more cane yield per unit of N fertilizer once the sugarcane plant has established using initial dose of nitrogen and thus AE₇₅ seems to be a more appropriate indicator for accounting N-nutrient use efficiency in sugarcane.     

Excess irradiance causes early symptoms of senescence during leaf expansion in photoautotrophically <Emphasis Type="Italic">in vitro</Emphasis> grown tobacco plants

Photosynthetic parameters, growth, and pigment contents were determined during expansion of the fourth leaf of in vitro photoautotrophically cultured Nicotiana tabacum L. plants at three irradiances [photosynthetically active radiation (400–700 nm): low, LI 60 μmol m⁻² s⁻¹; middle, MI 180 μmol m⁻² s⁻¹; and high, HI 270 μmol m⁻² s⁻¹]. During leaf expansion, several symptoms usually accompanying leaf senescence appeared very early in HI and then in MI plants. Symptoms of senescence in developing leaves were: decreasing chlorophyll (Chl) a+b content and Chl a/b ratio, decreasing both maximum (F_V/F_M) and actual (Φ_PS2) photochemical efficiency of photosystem 2, and increasing non-photochemical quenching. Nevertheless, net photosynthetic oxygen evolution rate (P _N) did not decrease consistently with decrease in Chl content, but exhibited a typical ontogenetic course with gradual increase. P _N reached its maximum before full leaf expansion and then tended to decline. Thus excess irradiance during in vitro cultivation did not cause early start of leaf senescence, but impaired photosynthetic performance and Chl content in leaves and changed their typical ontogenetic course.     

Long term exogenous putrescine application improves grain yield of a salt-sensitive rice cultivar exposed to NaCl

In order to determine the impact of polyamines on the yield-related parameters of rice (Oryza sativa L.) exposed to NaCl, the plants belonging to a salt-sensitive cultivar I Kong Pao were maintained from the young seedling stage until harvest on nutrient solutions containing 0 or 30 mM NaCl in the presence or absence of 10 μM putrescine (Put), 10 μM spermidine (Spd) or 10 μM spermine (Spm). Exogenous Put and to a lesser extent exogenous Spd improved growth and yield of salt-treated plants in relation to an increase in K⁺/Na⁺ ratio of shoots and roots as compared to plants exposed to NaCl in the absence of exogenous polyamines. Exogenous Put also improved the net CO₂ assimilation, at least partly as a consequence of an increase in the stomatal conductance. Yield increase of salt-treated plants exposed to Put was related to an improvement of floral morphogenesis leading to a higher number of fertile tillers per plant and a higher number of spikelets per panicle. Putrescine also improved the pollen viability in salt-treated plants, allowing a higher seed set and thus a higher grain yield per plant. Although polyamines accumulated in the shoots to some extent in response to exogenous application, neither Put nor Spd accumulated in the seeds. In contrast, Spm did not afford any protection of salt-treated plants but was translocated to the seeds during maturation. Seeds with a high internal Spm concentration exhibited delayed germination in the presence of NaCl. These data are discussed in relation to the implication of polyamine in the metabolism and physiology of salt-treated plants.     

Photosynthetic Response of Carrots to Varying Irradiances

Response to irradiance of leaf net photosynthetic rates (P _N) of four carrot cultivars: Cascade, Caro Choice (CC), Oranza, and Red Core Chantenay (RCC) were examined in a controlled environment. Gas exchange measurements were conducted at photosynthetic active radiation (PAR) from 100 to 1 000 μmol m⁻² s⁻¹ at 20 °C and 350 μmol (CO₂) mol⁻¹(air). The values of P _N were fitted to a rectangular hyperbolic nonlinear regression model. P _N for all cultivars increased similarly with increasing PAR but Cascade and Oranza generally had higher P _N than CC. None of the cultivars reached saturation at 1 000 μmol m⁻² s⁻¹. The predicted P _N at saturation (P _Nmax) for Cascade, CC, Oranza, and RCC were 19.78, 16.40, 19.79, and 18.11 μmol (CO₂) m⁻² s⁻¹, respectively. The compensation irradiance (I _c) occurred at 54 μmol m⁻² s⁻¹ for Cascade, 36 μmol m⁻² s⁻¹ for CC, 45 μmol m⁻² s⁻¹ for Oranza, and 25 μmol m⁻² s⁻¹ for RCC. The quantum yield among the cultivars ranged between 0.057–0.033 mol(CO₂) mol⁻¹(PAR) and did not differ. Dark respiration varied from 2.66 μmol m⁻² s⁻¹ for Cascade to 0.85 μmol m⁻² s⁻¹ for RCC. As P _N increased with PAR, intercellular CO₂ decreased in a non-linear manner. Increasing PAR increased stomatal conductance and transpiration rate to a peak between 600 and 800 μmol m⁻² s⁻¹ followed by a steep decline resulting in sharp increases in water use efficiency. This revised version was published online in August 2006 with corrections to the Cover Date.     

The influence of ultraviolet-B radiation on the growth, pigment production and chlorophyll fluorescence of Norway spruce seedlings

Norway spruce (Picea abies (L.)Karst.) from seven seed sources was grown in a greenhouse with 8.3 and 14.7 kJ·m⁻²·d⁻¹ m UV-B_BE (biologically effective UV-B: 280–320 nm) irradiation, and with no supplemental irradiation as control. The seedlings total biomass (dry weight) and shoot growth decreased with high UV-B treatment but spruce from low elevation seed sources were more affected. The seedlings grown at the highest UV-B irradiance (14.7 kJ·m⁻²·d⁻¹) showed from 5 to 38% inhibition of total biomass and 15 to 70 % shoot growth inhibition. Norway spruce populations from higher altitude seed sources manifested greater tolerance to UV-B radiation compared to plants from low altitudes. Changes in phospholipids and protective pigments were also determined. The plants grown at the lower UV-B irradiance (8.3 kJ·m⁻²·d⁻¹) showed greater ability to concentrations UV-B-absorbing pigments then control plants. Chlorophyll a fluorescence parameter R_fd, (R_fd=(F_m-F_s)/F_s) showed a significant decrease in needles of UV-B treated plants and this correlated with the altitude of seed source. Exposure to UV-B affect levels of the ratio of variable to maximum fluorescence (F_v/F_m). Results from this study suggest that the response to increased levels of UV-B radiation is depended upon the ecotypic differentiation of Norway spruce and involved changes in metabolites in plant tissues.     

Relationship between net photosynthesis and leaf respiration in Mediterranean evergreen species

The relationship between net photosynthetic (P _N) and leaf respiration (R) rates of Quercus ilex, Phillyrea latifolia, Myrtus communis, Arbutus unedo, and Cistus incanus was monitored in the period February 2006 to February 2007. The species investigated had low R and P _N during winter, increasing from March to May, when mean air temperature reached 19.2 °C. During the favourable period, C. incanus and A. unedo had a higher mean P _N (16.4±2.4 μmol m⁻² s⁻¹) than P. latifolia, Q. ilex, and M. communis (10.0±1.3 μmol m⁻² s⁻¹). The highest R (1.89±0.30 μmol m⁻² s⁻¹, mean of the species), associated to a significant P _N decrease (62 % of the maximum, mean value of the species), was measured in July (mean R/P _N ratio 0.447±0.091). Q₁₀, indicating the respiration sensitivity to short-term temperature increase, was in the range 1.49 to 2.21. Global change might modify R/P _N determining differences in dry matter accumulation among the species, and Q. ilex and P. latifolia might be the most favoured species by their ability to maintain sufficiently higher P _N and lower R during stress periods.