Water temperature instead of acclimation stage and oxygen concentration determines responses to winter floods

Field observations suggest that flooding events in the growing season are more detrimental than in winter. To clarify mechanisms producing these seasonal differences we analysed the role of plant acclimation, water temperature and oxygen concentration. We first tested the relative effects of seasonal acclimation and water temperature with three grassland species that differed in tolerance to summer floods (i.e. Rumex crispus, Rumex acetosa and Daucus carota). Our second experiment addressed the role of oxygen level relative to water temperature on biomass decay rate on a moderately intolerant species (i.e. R. acetosa).Irrespective of acclimation, biomass loss in warm water was considerably faster than in cold water. Given the concomitant decline in total non-structural carbohydrates, this was ascribed to the impact of water temperature on respiration rate. However, we only found a significant decline in carbohydrates for R. crispus and R. acetosa. D. carota seemed unable to access stored carbohydrates, which may explain its sensitivity for winter- and summer floods. Our second experiment provided no indication that the higher oxygen concentration may mitigate effects of flooding in cold water since a lower oxygen level of the water did not accelerate the rate of biomass loss.These findings indicate that temperature-driven respiration of carbohydrate reserves determines a species’ response to winter flooding, whereas oxygen level or plant acclimation are unimportant.     

Energy reserves during food deprivation and compensatory growth in juvenile roach: the importance of season and temperature

The effect of 21 days of starvation, followed by a period of compensatory growth during refeeding, was studied in juvenile roach Rutilus rutilus during winter and summer, at 4, 20 and 27° C acclimation temperature and at a constant photoperiod (12L : 12D). Although light conditions were the same during summer and winter experiments and fish were acclimated to the same temperatures, there were significant differences in a range of variables between summer and winter. Generally winter fish were better prepared to face starvation than summer fish, especially when acclimated at a realistic cold season water temperature of 4° C. In winter, the cold acclimated fish had a two to three‐fold larger relative liver size with an approximately double fractional lipid content, in comparison to summer animals at the same temperature. Their white muscle protein and glycogen concentration, but not their lipid content, were significantly higher. Season, independent of photoperiod or reproductive cycle, was therefore an important factor that determined the physiological status of the animal, and should generally be taken into account when fish are acclimated to different temperature regimes. There were no significant differences between seasons with respect to growth. Juvenile roach showed compensatory growth at all three acclimation temperatures with maximal rates of compensatory growth at 27° C. The replenishment of body energy stores, which were utilized during the starvation period, was responsible for the observed mass gain at 4° C. The contribution of the different energy resources (protein, glycogen and lipid) was dependent on acclimation temperature. In 20 and 27° C acclimated roach, the energetic needs during food deprivation were met by metabolizing white muscle energy stores. While the concentration of white muscle glycogen had decreased after the fasting period, the concentrations of white muscle lipid and protein remained more or less constant. The mobilization of protein and fat was revealed by the reduced size of the muscle after fasting, which was reflected in a decrease in condition factor. At 20° C, liver lipids and glycogen were mobilized, which caused a decrease both in the relative liver size and in the concentration of these substrates. Liver size was also decreased after fasting in the 4° C acclimated fish, but the substrate concentrations remained stable. This experimental group additionally utilized white muscle glycogen during food deprivation. Almost all measured variables were back at the control level within 7 days of refeeding.     

High cold tolerance through four seasons and all free-living stages in an ectoparasite

Off-host stages of temperate parasites must cope with low temperatures. Cold tolerance is often highest in winter, as a result of diapause and cold acclimation, and low during the active summer stages. In some blood-feeding ectoparasites, offspring provisioning determines cold tolerance through all the non-feeding, off-host stages. Large size increases survival in the cold, but so far seasonal variation in within-female offspring size has not been associated with offspring cold tolerance. The deer ked (Lipoptena cervi) reproduces on cervids from autumn to spring. Newborn pupae drop off the host, facing frosts without any acclimation. We examined cold tolerance through 4 seasons and from birth to adulthood by means of short- and long-term frost exposure. We expected females to produce more tolerant offspring in winter than in spring. Large spring pupae survived prolonged frosts better than did small winter pupae. Thus more tolerant offspring were not produced when the temperature outside the host is at its lowest. Unexpectedly, the freezing points were -20 °C or below all year round. We showed that high cold tolerance is possible without acclimation regardless of life stage, which presumably correlates with other survival characteristics, such as the starvation resistance of free-living ectoparasites.     

Change in antioxidant responses against oxidative damage in black chickpea following cold acclimation

Cold stress is an important factor affecting chickpea (Cicer arietinum L.) plants in winter and early spring. We evaluated the effects of cold stress by measuring lipid peroxidation, membrane permeability, and some enzyme activities involved in the ROS-scavenging system under acclimation and non-acclimation conditions in black chickpea Kaka, a popular genotype planted, and accession 4322, as a landrace genotype. Under non-acclimation conditions, the genotype 4322 prevented the H₂O₂ accumulation more efficiently, which led to a decrease in lipid peroxidation and membrane permeability compared to Kaka. Studying the activities of antioxidant enzymes showed that catalase was more effective enzyme in cell protection against H₂O₂ in 4322 plants. Such response in acclimated plants was more pronounced than in control and nonacclimated plants. In this study, the increase in guaiacol peroxidase and ascorbate peroxidase activities did not preserve cell membranes from oxidative damage in Kaka plants. It was observed that short-term acclimation can induce greater cold tolerance upon the increase of oxidative stress in chickpea plants. This was due to low levels of MDA and electrolyte leakage index, indicating the lower lipid peroxidation and higher membrane stability under the cold stress compared to non-acclimated plants.     

The activity of pro- and antioxidant systems in the liver of freshwater fishes during different seasons

The content of lipid peroxidation products--diene conjugates, lipid hydroperoxides, thiobarbituric acid reactive substances (TBARS), vitamins A, E and carotenoids and the activity of antioxidant enzymes--superoxide dismutase, glutathione peroxidase and catalase in the liver of freshwater fishes of different species (silver carp, grass carp and common carp) in different seasons have been studied. It was established the activity of antioxidant defence system in the liver of fish depends significantly on the season and fish species. In particular, the content of lipid peroxidation products in the liver of freshwater fishes at the beginning of winter and spring was significantly higher compared to their content at the beginning of summer and autumn. The superoxide dismutase and glutathione peroxidase activities in the liver of these fish species at the beginning of winter and spring were significantly lower than at the beginning of summer and autumn while the seasonal changes of catalase activity in the liver of fish are expressed insignificantly. The content of vitamins E, A1, A2 and carotenoids in the liver of fishes of different species at the beginning of winter and spring was significantly lower than at the beginning of summer and autumn. The content of lipid peroxidation products and vitamins E, A1 and A2 in the liver of common carp is significantly lower than in the liver of silver carp and grass carp and species differences in antioxidant enzymes activity are insignificant.     

The penalty of a long,hot summer. Photosynthetic acclimation to high CO2 and continuous light in "living fossil" conifers

Deciduous forests covered the ice-free polar regions 280 to 40 million years ago under warm "greenhouse" climates and high atmospheric pCO2. Their deciduous habit is frequently interpreted as an adaptation for minimizing carbon losses during winter, but experiments with "living fossils" in a simulated warm polar environment refute this explanation. Measured carbon losses through leaf abscission of deciduous trees are significantly greater than losses through winter respiration in evergreens, yet annual rates of primary productivity are similar in all species. Here, we investigate mechanisms underlying this apparent paradox by measuring the seasonal patterns of leaf photosynthesis (A) under pCO2 enrichment in the same trees. During spring, A increased significantly in coastal redwood (Sequoia sempervirens), dawn redwood (Metasequoia glyptostroboides), and swamp cypress (Taxodium distichum) at an elevated pCO2 of 80 Pa compared with controls at 40 Pa. However, strong acclimation in Rubisco carboxylation capacity (Vc,max) completely offset the CO2 response of A in all species by the end of 6 weeks of continuous illumination in the simulated polar summer. Further measurements demonstrated the temporary nature of acclimation, with increases in Vc,max during autumn restoring the CO2 sensitivity of A. Contrary to expectations, the acclimation of Vc,max was not always accompanied by accumulation of leaf carbohydrates, but was associated with a decline in leaf nitrogen in summer, suggesting an alteration of the balance in plant sources and sinks for carbon and nitrogen. Preliminary calculations using A indicated that winter carbon losses through deciduous leaf abscission and respiration were recovered by 10 to 25 d of canopy carbon fixation during summer, thereby explaining the productivity paradox.     

Leaves of Field-Grown Mastic Trees Suffer Oxidative Stress at the Two Extremes of their Lifespan

Leaf senescence is a complex phenomenon occurring in all plant species, but it is still poorly understood in plants grown in Mediterranean field conditions and well-adapted to harsh climatic conditions. To better understand the physiological processes underlying leaf senescence in mastic trees (Pistacia lentiscus L.), we evaluated leaf growth, water and N content, photosystem II (PSII) photochemistry, lipid peroxidation and levels of photosynthetic pigments, antioxidants, abscisic acid, and salicylic acid and jasmonic acid during the complete leaf lifespan, from early expansion to late senescence in relation to natural climatic conditions in the field. While mature leaves suffered from water and N deficit during late spring and summer, both young (emerging) and old (senescing) leaves were most sensitive to photo-oxidative stress, as indicated by reductions in the F v/F m ratio and enhanced lipid peroxidation during late autumn and winter. Reductions in the F v/F m ratio were associated with low α-tocopherol (vitamin E) levels, while very old, senescing leaves additionally showed severe anthocyanin losses. We have concluded that both young (emerging) and old (senescing) leaves suffer oxidative stress in mastic trees, which may be linked in part to suboptimal temperatures during late autumn and winter as well as to low vitamin E levels.     

Seasonal pattern of antioxidant enzyme system in the roots of perennial forage grasses grown in alpine habitat, related to freezing tolerance

The relationship between active oxygen species (AOS) and membrane damage, and between antioxidant enzyme activity and chilling tolerance has been documented, but the mechanisms responsible for perennial forage grass to survive winter with temperatures at ?30°C in temperate alpine regions is not well understood. In this study, the seasonal pattern of enzymatic antioxidant systems superoxide dismutase (EC 1.15.1.1), catalase (EC 1.11.1.6), peroxidase (EC 1.11.1.7), ascorbate peroxidase (EC 1.11.1.11) and lipid peroxidation in roots and leaves of alpine perennial grasses grown in their natural environment were investigated to understand the role of the enzymatic antioxidant system in freezing tolerance of perennial grasses. Four grasses, Poa sphyondylodes Trine., Bromus inermis Leyss., Bromus sinensis Keng. and Elymus nutans Griseb., were established in alpine conditions in 1993. The grasses were sampled at approximately semi‐monthly intervals in the autumn of 1995 and spring of 1996. The results showed that leaves were dead in the autumn and membrane damage seems to play a key role in the decline of this organ. Antioxidant enzyme activities of the roots strongly changed with declining temperature in the autumn and winter or increasing temperature in the spring. With the decrease in temperature in the autumn the antioxidant enzyme activities increased rapidly, reaching maximum values in early November and then slowly declining during the following winter period, although they were still higher than in September In the spring, antioxidant enzymes activities increased again in the roots with the rise of temperature from mid April to early May when the shoots began re‐growth. In contrast, thiobarbituric acid‐reactive substances content in the roots increased markedly in the autumn, reaching maximum values in early October and remaining constant with little fluctuation during the following winter. In the autumn when the roots experienced winter acclimation, the formation of freezing tolerance in the roots was correlated with the activities of the antioxidant enzyme, indicating that antioxidant activity systems in the roots played an important role in limiting the production of free radicals to protect membrane integrity. Freezing tolerance in alpine grasses correlated with an increased capacity to scavenge or detoxify activated AOS by the antioxidant enzymatic system. AOS accumulated with decreasing temperature in early cold acclimation may be an inducer in activating the antioxidant enzyme defence system for the formation of freezing tolerance in roots.     

Seasonal changes in markers of oxidative damage to lipids and DNA; correlations with seasonal variation in diet

We have addressed the question whether the relatively high incidence of cardiovascular disease and certain cancers in countries of central/eastern Europe might be associated with nutritional imbalance, in particular a lack of fresh fruit and vegetables in the diet in winter months. Nutritional parameters and markers of oxidative stress were studied in three Slovak population groups: 46 survivors of myocardial infarction (MI group) and 48 healthy, normolipidemic subjects (NL), living in or near Bratislava; and 70 rural controls (RC group) living a more traditional life style in a country town. Data were collected in February/March and September/October of two consecutive years, representing times of minimum and maximum local availability of fresh fruits and vegetables. Oxidative stress was monitored using two biomarkers; plasma malondialdehyde (MDA, a product of lipid peroxidation), and oxidation of lymphocyte DNA. Dietary antioxidants, folic acid, homocysteine, total antioxidant status (FRAP) and uric acid were measured in plasma. Food frequency questionnaires were administered. Vegetable consumption in summer/autumn was twice as high as in winter/spring. DNA damage did not vary consistently across the seasons. Mean plasma MDA levels for the MI and NL groups showed a clear pattern, with high levels in winter/spring and low levels in summer/autumn. Folic acid showed a reciprocal pattern, similar to the pattern of vegetable consumption. The RC group had the smallest seasonal variations in vegetable consumption, folic acid levels, and MDA. High winter MDA levels are seen in those individuals with relatively low folic acid; they never occur in subjects with high plasma folic acid, implying that folic acid might directly protect against lipid oxidation. This study illustrates the value of the molecular epidemiological approach, while emphasising the need for well characterised population groups and valid biomarkers.     

Cold hardiness in summer and winter diapause and post-diapause pupae of the cabbage armyworm, Mamestra brassicae L. under temperature acclimation

Cold hardiness and biochemical changes were investigated in winter and summer pupae of the cabbage armyworm Mamestra brassicae at the diapause and post-diapause stages under temperature acclimation. Diapause pupae were successively acclimated to 25, 20 and then 10 degrees C (warm-acclimated group). Pupae at the diapause and post-diapause stages were successively acclimated to 5, 0, -5 and then -10 degrees C (cold-acclimated groups). Supercooling point values in winter and summer pupae remained constant regardless of the diapause stages and acclimated temperatures. Warm-acclimated pupae at the diapause stage did not survive the subzero temperature exposure, whereas, cold-acclimated pupae achieved cold hardiness to various degrees. Winter pupae were more cold hardy than summer pupae, and pupae at the post-diapause stage were more cold hardy than those at the diapause stage. Trehalose contents in winter pupae rose under cold acclimation. Summer pupae accumulated far lower trehalose contents than winter pupae, with the maximal level occurring in winter pupae at the post-diapause stage. Glycogen content remained at a high level in diapause pupae after warm acclimation, whereas it decreased after cold acclimation. Alanine, the main free amino acid in haemolymph after cold acclimation, increased at lower temperatures in both diapause and post-diapause pupae, but the increase was greater in the diapause pupae. These results suggest that cold hardiness is more fully developed in winter pupae than in summer pupae, and cold acclimation provides higher cold hardiness in winter pupae at the post-diapause stage than at the diapause stage.     

Phenolic compounds in Norway spruce as affected by boron nutrition at the end of the growing season

The increase in concentrations of phenolic compounds in boron (B) deficiency has been demonstrated in many herbaceous plant species, but information on woody plants is scarce. It has been suggested that accumulation of phenolic compounds plays a role in the development of cold hardiness in herbaceous plants but also that B deficiency decreases winter hardiness. Here we study the effects of B nutrition on phenolic compounds in Norway spruce (Picea abies L.) in the course of cold acclimation. Phenolic compounds were analysed in Norway spruce seedlings from three different B-fertilisation treatments in two harvests: non-acclimated and cold-acclimated seedlings. Norway spruce phenolic compounds consisted mainly of condensed tannins. During B deficiency, condensed tannins and monocoumaroyl–astragalin der. 1 increased in non-acclimated seedlings. The increase in tannins was 21%, which was nearly significant. However, the effect of B on phenolic compounds was almost absent in cold-acclimated seedlings. The condensed tannin concentration increased much more with time in the simulated autumn than due to B deficiency, and we conclude that the B effect was probably not large enough to be important for the hardening of the seedlings. The total phenolic concentrations more than doubled during the course of cold hardening suggesting that phenolics have a role in the winter hardiness in Norway spruce.     

Seasonal Changes of the Metabolites in the Leaves, Bark and Xylem Tissues of Olive Tree (Olea europaea. L) II. Carbohydrates

Seasonal changes of starch and soluble carbohydrates in leaves,bark and xylem tissues of olive tree were examined during acomplete annual cycle. Leaf starch and soluble carbohydrateswere detected at high levels during the spring and autumn metabolicallyactive periods. The low level of leaf starch in combinationwith the drastic reduction of soluble carbohydrates and mannitol,defined the summer period of the low metabolic state of thetree. The low leaf starch level in conjunction with the risensoluble carbohydrate levels in leaves in winter were associatedwith cold acclimation processes. The bark and xylem tissueswere performing as starch deposition sites, and differencesin the extent of starch accumulation in these tissues were detectedduring the seasons. The starch fluctuations in bark and xylemwere discussed in relation to the translocation of metabolitesand other physiological processes. Mannitol, the most abundantleaf carbohydrate, was examined in relation to the reducingsugars exported to the bark. The bark mannitol was examinedin conjunction with the sucrose, glucose and starch levels inthe maturing bark tissues and was correlated to the low wintertemperatures. During the winter there was a drastic reductionof mannitol circulation from the bark to xylem. Olive tree, Olea europaea, L, carbohydrates     

高寒山区牧草根中丙二醛、渗透调节物、多胺季节动态与抗冻力关系研究

温带地区的高山多年生草本植物可在－３０℃组织结冰状况下生存,然而人们并不了解其抗冻的生理机理。本研究目的拟通过测定自然生境下生长的４种高寒山区禾本科牧草（无芒雀麦（Ｂｒｏｍｕｓ ｉｎｅｒｍｉｓ Ｌｅｙｓｓ．）、花雀麦（Ｂｒｏｍｕｓ　 ｓｉｎｅｎｓｉｓ　 Ｋｅｎｇ．）、垂穗披碱草（Ｅｌｙｍｕｓ ｎｕｔａｎｓ Ｇｒｉｓｅｂ．）、草地早熟禾（Ｐｏａ ｓｐｈｙｏｎｄｙｌｏｄｅｓ　 Ｔｒｉｎ．）根中渗透调节物、膜脂过氧化产物、多胺含量季节变化,以了解他们与牧草抗冻的关系。结果表明,在晚秋（９月１～１５日）牧草根中ＭＤＡ含量增高,尔后下降,冬季保持恒定。总碳水化合物（ＴＮＣ）,可溶性糖、脯氨酸、可溶性蛋白质含量随晚秋气温下降而增加,在１１月达到最高,尔后下降,且持续到翌年春季。随晚秋气温下降从９月到１１月根中多胺含量迅速增加,４种牧草平均增加１８０％,其中亚精胺（Ｓｐｄ）占多胺含量的５３％。在１１月牧草根中多胺几乎完全消失。上述物质在晚秋入冬增加正好与牧草抗冻锻炼时间相吻合,因而是植物抗冻适应的重要生理响应和植物越冬的低温保护物质。他们在降低细胞冰点、防止细胞结冰引起的膜机械伤害,抑制膜脂过氧化保护膜稳定性方面具有重要作用。     

Food reserves of scots pine (Pinus sylvestris L.)

Summary Starch, soluble sugars, triacylglycerols, diacylglycerols and free fatty acids were measured in 30-year-old Scots pine (Pinus sylvestris L.) trees during an annual cycle in the sapwood (youngest ten xylem rings). The radial distribution of carbohydrates and lipids was studied in the trunkwood of 90 -to 150-year-old Scots pine trees collected at the end of the growing season. Determination of the compounds was performed using specific enzymatic assays, capillary gas chromatography and thin layer chromatography. The amounts of glucose, fructose, sucrose, and galactose/arabinose in the sapwood were slightly higher in winter than in summer. Raffinose/stachyose increased up to 5-fold during the cold period. At the beginning of the growing season starch amounts rose, and then decreased in summer and autumn. No concentration changes were observed in the total amounts of diacylglycerols and fatty acids throughout the year. Triacylglycerol levels were slightly higher in February than in summer and autumn. Relative frequencies of individual fatty acids were similar in all lipid fractions. Glucose, fructose, sucrose, starch and triacylglycerols disappeared almost entirely at the transition zone from sapwood to heartwood. In contrast, free fatty acids and galactose/arabinose rose in centripetal direction, and diacylglycerols remained constant across trunk cross-sections. The relative amounts of individual fatty acids changed markedly in the free fatty acid fraction and in the triacylglycerols when crossing the sapwood-heartwood boundary. Concentration changes of reserve materials are discussed in relation to season, mobilization and translocation processes, dormancy, frost resistance, and heartwood formation. The results are compared to those found in needles.     

Ecotype-dependent control of growth,dormancy and freezing tolerance under seasonal changes in <Emphasis Type="Italic">Betula pendula</Emphasis> Roth

Woody plants in the temperate and boreal zone undergo annual cycle of growth and dormancy under seasonal changes. Growth cessation and dormancy induction in autumn are prerequisites for the development of substantial cold hardiness in winter. During evolution, woody plants have developed different ecotypes that are closely adapted to the local climatic conditions. In this study, we employed distinct photoperiodic ecotypes of silver birch (Betula pendula Roth) to elucidate differences in these adaptive responses under seasonal changes. In all ecotypes, short day photoperiod (SD) initiated growth cessation and dormancy development, and induced cold acclimation. Subsequent low temperature (LT) exposure significantly enhanced freezing tolerance but removed bud dormancy. Our results suggested that dormancy and freezing tolerance might partially overlap under SD, but these two processes were regulated by different mechanisms and pathways under LT. Endogenous abscisic acid (ABA) levels were also altered under seasonal changes; the ABA level was low during the growing season, then increased in autumn, and decreased in winter. Compared with the southern ecotype, the northern ecotype was more responsive to seasonal changes, resulting in earlier growth cessation, cold acclimation and dormancy development in autumn, higher freezing tolerance and faster dormancy release in winter, and earlier bud flush and growth initiation in spring. In addition, although there was no significant ecotypic difference in ABA level during growing season, the rates and degrees of ABA alterations were different between the ecotypes in autumn and winter, and could be related to ecotypic differences in dormancy and freezing tolerance.     

Annual changes in the concentration of minerals and organic compounds of Quercus suber leaves

Mature leaves are the primary source of sugars, which give rise to many secondary metabolites required for plant survival under adverse conditions. In order to study the interaction of field‐grown cork oak (Quercus suber L.) with the environment, we investigated the seasonal variation of minerals and organic metabolites in the leaves, using inductively coupled plasma atomic emission spectrometry, elemental analysis and nuclear magnetic resonance spectrometry. Statistical analysis showed that the data strongly correlated with seasonal climate and were divided in three groups corresponding to: (1) spring‐early summer, (2) summer and (3) autumn‐winter. The concentration of N, P, K and leaf ash content were highest in spring (recently formed leaves), reached the minimum during the hot and dry summer and increased slightly during the rainy period of autumn‐winter. Conversely, Na, Mg and Ca concentrations were lowest in spring‐early summer and increased during summer and autumn‐winter, the Ca concentration increasing five‐fold. Two cyclitol derivatives, quinic acid and quercitol were the major organic metabolites of the leaves. Their concentration along the season followed opposite trends. While quinic acid predominated during spring‐early summer, when it contributed 12% to the leaf osmotic potential, quercitol was predominant during autumn‐winter, when its contribution to leaf osmotic potential was about 10%. This different preponderance of the two compounds is expressed by the quercitol/quinic acid ratio, which can be as low as 0.2 in early summer and as high as 9 in winter. Sucrose and glucose concentrations also increased during autumn‐winter. Evidence for the quercitol protective role in plants during stress is discussed, and on the basis of structural similarity, it is suggested that quinic acid could have an identical importance, with a protective role against heat and high irradiance. It is concluded that the marked changes in Q. suber leaf composition throughout the year could have important implications in the plant capacity to endure climatic stress.     

Hydrogen peroxide pre-treatment induces salt-stress acclimation in maize plants

The effect of exogenously applied H2O2 on salt stress acclimation was studied with regard to plant growth, lipid peroxidation, and activity of antioxidative enzymes in leaves and roots of a salt-sensitive maize genotype. Pre-treatment by addition of 1 microM H2O2 to the hydroponic solution for 2 days induced an increase in salt tolerance during subsequent exposure to salt stress. This was evidenced by plant growth, lipid peroxidation and antioxidative enzymes measurements. In both leaves and roots the variations in lipid peroxidation and antioxidative enzymes (superoxide dismutase, ascorbate peroxidase, guaiacol peroxidase, glutathione reductase, and catalase) activities of both acclimated and unacclimated plants, suggest that differences in the antioxidative enzyme activities may, at least in part, explain the increased tolerance of acclimated plants to salt stress, and that H2O2 metabolism is involved as signal in the processes of maize salt acclimation.     

Improving the thiobarbituric acid-reactive-substances assay for estimating lipid peroxidation in plant tissues containing anthocyanin and other interfering compounds

The occurrence of malondialdehyde (MDA), a secondary end product of the oxidation of polyunsaturated fatty acids, is considered a useful index of general lipid peroxidation. A common method for measuring MDA, referred to as the thiobarbituric acid-reactive-substances (TBARS) assay, is to react it with thiobarbituric acid (TBA) and record the absorbance at 532 nm. However, many plants contain interfering compounds that also absorb at 532 nm, leading to overestimation of MDA values. Extracts of plant tissues including purple eggplant (Solanum melongena L.) fruit, carrot (Daucuscarota L.) roots, and spinach (Spinacia oleracea L.) leaves were assessed for the presence of MDA and other non-MDA compounds absorbing at 532 nm. A method described herein corrects for these interferences by subtracting the absorbance at 532 nm of a solution containing plant extract incubated without TBA from an identical solution containing TBA. The reliability and efficiency of this spectrophotometric method was assessed by altering the relative ratios of exogenous MDA additions and/or extracts of red cabbage (Brassica oleracea L.) leaves containing interfering compounds and then measuring MDA recovery. Reliability was also validated through high-performance liquid chromatography and high-performance liquid chromatography-mass spectrometry techniques. Results indicated that over 90% of exogenously added MDA could be recovered through the improved protocol. If there were no corrections for interfering compounds, MDA equivalents were overestimated by up to 96.5%. Interfering compounds were not detected in vegetables such as lettuce (Lactuca sativa L.) and spinach which had low or negligible concentrations of anthocyanidin derivatives. Comparisons between the TBARS method presented here and two currently accepted protocols indicated that the new modified method exhibits greater accuracy for quantifying TBA-MDA levels in tissues containing anthocyanins and/or other interfering compounds. This modified protocol represents a facile and rapid method for assessment of lipid peroxidation in virtually all plant species that contain interfering compounds. Received: 28 August 1998 / Accepted: 29 September 1998