Development of antioxidative defense system of wheat seedlings in response to high light

Changes in the activities of enzymes involved in the detoxification of reactive oxygen species in wheat seedlings ( Triticum aestivum L.) in response to variations in the light environment were studied. Activities of ascorbate peroxidase, superoxide dismutase, monodehydroascorbate reductase, dehydroascorbate reductase, glutalhione reductase and catalase were much lower in seedlings grown under low-light conditions than in those grown under high-light conditions. Activities of all these enzymes significantly increased within 24 h of transfer of the low-light-grown seedlings to the high-light regime. The results suggest that the increase in enzyme activities was an adaptive response of the plants to higher amounts of active oxygen species generated at higher light intensities. An accumulation of glutathione was also observed, which could also be a part of the defense strategy to meet the increased generation of active oxygen species upon transfer of low-light-grown plants to high-light conditions.     

Modulation of ethylene synthesis in acotyledonous soybean and wheat seedlings

The characteristics of ethylene production and ACC conversion in 8-day-old soybean seedlings were examined and a relationship between cytochrome P-450 activity and ethylene-forming enzyme (EFE) activity was found. An atmosphere containing 10% carbon monoxide (CO) significantly inhibited ethylene production and ACC conversion in control soybean seedlings, but had only a slight effect on soybean seedlings treated with uniconazole. Foliar application of triclopyr, a pyridine analogue of the phenoxy herbicides, significantly increased ethylene production and ACC conversion in control, but not in uniconazoletreated seedlings. Triclopyr treatment also resulted in a three-fold increase in extractable cytochrome P-450 of 5-day-old etiolated soybeans. At equimolar concentrations tetcyclacis was more effective than uniconazole in reducing shoot elongation and endogenous ethylene production. Although uniconazole and tetcyclacis did not inhibit ACC conversion in nonherbicide-treated soybean seedlings, they did prevent the observed increase in ACC-dependent EFE activity following triclopyr application. However, the rate of ACC conversion in etiolated soybean segments was sensitive to uniconazole, and tetcyclacis inhibited the rate of ACC conversion by 2.6-fold in etiolated soybean segments within 4 h after treatment. Microsomal membranes were isolated from 5-day-old naphthalic anhydride-treated etiolated wheat shoots as this tissue contains much higher cytochrome P-450 levels than soybean shoots. Optical difference spectroscopy demonstrated that ACC generated binding spectrum characteristic of a reverse-type-I cytochrome P-450 substrate when combined with reduced microsomes. In vitro conversion of ACC to ethylene by microsomal membranes was NADPH-dependent, inhibited by CO, and had an apparent K_m and V_max of 45 M and 0.345 nl/mg protein/h, respectively. These results suggest that cytochrome P-450-mediated monooxygenase reactions may be intimately involved in the conversion of ACC to ethylene in young soybean and wheat seedlings.     

Mode of high temperature injury to wheat during grain development

High temperature stress adversely affects wheat growth in many important production regions, but the mode of injury is unclear. Wheat ( Triticum aestivum L. cv. Newton) was grown under controlled conditions to determine the relative magnitude and sequences of responses of source and sink processes to high temperature stress during grain development. Regimes of 25°C day/15°C night, 30°C day/20°C night, and 35°C day/25°C night from 5 days after anthesis to maturity differentially affected source and sink processes. High temperatures accelerated the normal decline in viable leaf blade area and photosynthetic activities per unit leaf area. Electron transport, as measured by Hill reaction activity, declined earlier and faster than other photosynthetic processes at the optimum temperature of 25/15 °C and at elevated temperatures. Changes in RUBP carboxylase activities were similar in direction but smaller in magnitude than changes in photosynthesic rate. Increased protease activity during senscence was markedly accentuated by high temperature stress. Specific protease activity increased 4-fold at 25/15 °C and 28-fold at 35/25 °C from 0 to 21 days after initiation of temperature treatments. Grain-filling rate decreased from the lowest to the highest temperature, but the change was smaller than the decrease in grain-filling duration at the same temperatures. We concluded that a major effect of high temperature is acceleration of senescence, including cessation of vegetative and reproductive growth, deterioration of photosynthetic activities, and degradation of proteinaceous constituents.     

Nitrogen assimilation and protein synthesis in wheat seedlings as affected by mineral nutrition. I. Macronutrients

Deficiencies of each macronutrient (N, P, K, Ca. Mg, S, and Fe) decreased the specific activity of nitrate reductase from Triticum aestivum L. seedlings. Nitrate content was decreased by N, P, K, Ca, and Mg deficiencies and unaffected by S and Fe deficiencies. Glutamic acid dehydrogenase activity was decreased by N, P, and S deficiencies, unchanged by K deficiency, and increased by Ca, Mg, and Fe deficiencies. Glutamine synthetase activity closely paralleled nitrate reductase activity and was decreased by deficiencies of N, P, K, Ca, Mg, and S. Glutamic-oxaloacetic transaminase was not sensitive to macronutrient deficiencies. High ¹⁴C-leucine incorporation into tissue sections of N-, P-, K-, Ca-, and S-deficient seedlings did not appear indicative of protein synthesis rates in intact seedlings. Nutritional deficiencies apparently depleted endogenous amino acid pools and caused less inhibition of exogenous ¹⁴C-leucine incorporation into protein.     

A quick method to screen high and low yielding wheat cultivars exposed to high temperature

In tropical countries, high temperature stress is the major abiotic stress, which controls the productivity and yield of crop plants. Two high yielding and low yielding genotypes of durum wheat were selected for detailed analysis of their photochemical efficiencies. In low yielding genotypes (Malvi local and Sawer local), the whole primary photochemical reactions are affected before and after heat stress. The results show that low yielding genotypes show less efficiency in the usage of the available excitation energy. This is a case study to establish use of chlorophyll a fluorescence measurement as an effective tool to screen plants for their stress tolerance. The study is important for stress physiology and may be useful for assessment of stress tolerant plants.     

Influence of temperature increase on evaportranspiration rate and cytokinin content in wheat seedlings

The content of cytokinins especially zeatin nucleotide decreased in shoots as a result of temperature increase. Simultaneously the cytokinins accumulated in roots. The changes in cytokinins distribution were followed by a decline of evapotranspiration after its initial temperature-induced uprising. This revised version was published online in July 2006 with corrections to the Cover Date.     

Presence of an alpha-amylase isozyme with high temperature optima in the wheat variety tolerant to high temperature at juvenile plant stage

In the present study α-amylase was partially purified from detached grains of five day old seedlings of two wheat (Triticum aestivum L.) varieties, showing differential responses to high temperature stress at seedling stage. A three step purification via ammonium sulphate precipitation, DEAE-cellulose column chromatography and gel filtration on Sephadex G-150 was employed. A single α-amylase was detected in the high temperature sensitive PBW-175 variety, while two isozymes namely, α-amylase-1 and α-amylase-2 were obtained in the relatively tolerant WL-711 variety. The pH optima of the three α-amylases were in 5.0–5.5 range and comparable to the cereal amylases. The temperature optima of PBW-175 α-amylase and α-amylase-1 of WL-711, which appeared to be the major isozyme of the variety, were same at 45 °C and also comparable to cereal amylases. On the other hand the optimum temperature for α-amylase-2 was high at 70 °C, which is unusual and not reported earlier for cereal amylases. The Km of PBW-175 α-amylase was lower than the Km values of WL-711 isozymes, this was well co-related with an overall high α-amylase activity detected in the detached grains of five day old seedlings of PBW-175 compared to WL-711. However WL-711 variety showed a better inherent seedling growth, vigour and EUE than PBW-175, may be because it had two α-amylase isozymes which could compensate for the higher enzyme activity detected in PBW-175. Moreover, the presence of α-amylase-2 in the grain of WL-711 having temperature optima of 70 °C, possibly rendered its seedlings tolerant to HS of 50 °C, while the seedlings of PBW-175 succumbed to this temperature shock.     

Effects of exogenous spermidine on antioxidant system of tomato seedlings exposed to high temperature stress

The effects of foliar spraying with spermidine (Spd) on antioxidant system in tomato (Lycopersicon esculentum Mill.) seedlings were investigated under high temperature stress. The high temperature stress significantly inhibited plant growth and reduced chlorophyll (Chl) content. Application of exogenous 1 mM Spd alleviated the inhibition of growth induced by the high temperature stress. Malondialdehyde (MDA), hydrogen peroxide (H₂O₂) content and superoxide anion (O₂) generation rate were significantly increased by the high temperature stress, but Spd significantly reduced the accumulation of reactive oxygen species (ROS) and MDA content under the stress. The high temperature stress significantly decreased glutathione (GSH) content and activities of superoxide dismutase (SOD), peroxidase (POD), catalase (CAT), ascorbate peroxidase (APX), glutathione reductase (GR), monodehydroascorbate reductase (MDHAR) and dehydroascorbate reductase (DHAR), but increased contents of dehydroascorbic acid (DHA), ascorbic acid (AsA), and oxidized glutathione (GSSG) in tomato leaves. However, Spd significantly increased the activities of antioxidant enzymes, levels of antioxidants and endogenous polyamines in tomato leaves under the high temperature stress. In addition, to varying degrees, Spd regulated expression of MnSOD, POD, APX2, APX6, GR, MDHAR, DHAR1, and DHAR2 genes in tomato leaves exposed to the high temperature stress. These results suggest that Spd could change endogenous polyamine levels and alleviate the damage by oxidative stress enhancing the non-enzymatic and enzymatic antioxidant system and the related gene expression.     

Heat shock protein synthesis and thermal tolerance in wheat

Plants respond to high temperature stress by the synthesis of an assortment of heat shock proteins that have been correlated with an acquired thermal tolerance to otherwise lethal temperatures. This study was conducted to determine whether genotypic differences in acquired thermal tolerance were associated with changes in the pattern of heat shock protein synthesis. The pattern of heat shock protein synthesis was analyzed by ³⁵S-methionine incorporation in wheat (Triticum aestivum L.) varieties exhibiting distinct levels of acquired thermal tolerance. Significant quantitative differences between the cultivars Mustang and Sturdy were observed in the HSP exhibiting apparent molecular weights of 16, 17, 22, 26, 33, and 42 Kilodaltons. Genotypic differences in the synthesis of the small subunit of ribulose 1,5-bisphosphate carboxylase/oxygenase were observed at 34°C. Two-dimensional electrophoretic analysis revealed unique proteins (16, 17, and 26 kilodaltons) in the thermal tolerant variety Mustang that were absent in the more thermal sensitive variety Sturdy. These results provide a correlation between the synthesis of specific low molecular weight heat shock proteins and the degree of thermal tolerance expressed following exposure to elevated temperatures.     

Characterization of high temperature induced stress impairments in thylakoids of rice seedlings.

Exposure of isolated thylakoids or intact plants to elevated temperature is known to inhibit photosynthesis at multiple sites. We have investigated the effect of elevated temperature (40 degrees C) for 24 hr in dark on rice seedlings to characterize the extent of damage by in vivo heat stress on photofunctions of photosystem II (PSII). Chl a fluorescence transient analysis in the intact rice leaves indicated a loss in PSII photochemistry (Fv) and an associated loss in the number of functional PSII units. Thylakoids isolated from rice seedlings exposed to mild heat stress exhibited >50% reduction in PSII catalyzed oxygen evolution activity compared to the corresponding control thylakoids. The ability of thylakoid membranes from heat exposed seedlings to photooxidize artificial PSII electron donor, DPC, subsequent to washing the thylakoids with alkaline Tris or NH2OH was also reduced by approximately 40% compared to control Tris or NH2OH washed thylakoids. This clearly indicated that besides the disruption of oxygen evolving complex (OEC) by 40 degrees C heat exposure for 24 hr, the PSII reaction centers were impaired by in vivo heat stress. The analysis of Mn and manganese stabilizing protein (MSP) contents showed no breakdown of 33 kDa extrinsic MSP and only a marginal loss in Mn. Thus, we suggest that the extent of heat induced loss of OEC must be due to disorganization of the OEC complex by in vivo heat stress. Studies with inhibitors like DCMU and atrazine clearly indicated that in vivo heat stress altered the acceptor side significantly. [14C] Atrazine binding studies clearly demonstrated that there is a significant alteration in the QB binding site on D1 as well as altered QA to QB equilibrium. Thus, our results show that the loss in PSII photochemistry by in vivo heat exposure not only alters the donor side but significantly alters the acceptor side of PSII.     

Action of light and streptomycin on protein synthesis in cabbage seedlings

The action of light on protein synthesis was examined in the cabbage seedlings, a system extensively used in the studies of anthocyanin synthesis. Continuous red and far red light have no effect on total protein content while they cause a marked decrease in the level of free amino acids in cabbage seedlings. The rate of protein synthesis, measured as incorporation of radioaetively-labelled amino acids into proteins, is clearly stimulated by light. Phytochrome involvement in the light stimulation of the incorporation is also demonstrated by the red-far red reversibility of the response. The relative effectiveness of continuous red and far red light upon the incorporation of amino acids into proteins is affected by the nature of the system used to study the incorporation process. When excised cotyledons and short period of incorporation were used, continuous far red was more effective than red. However, when whole seedlings and long period of incorporation were used, red and far red were equally effective. Streptomycin causes a 10– 15% decrease in the rate of incorporation of amino acids into proteins of all cellular fractions, except the plastid fraction where a much higher inhibition (30%) was observed.