Monitoring Primary Response to Chilling Stress in Etiolated Vigna radiata and V. mungo Seedlings Using Thermal Hysteresis of Water Proton NMR Relaxation Times

Thermal hysteresis of longitudinal relaxation times (T₁) ofwater protons in hypocotyls of etiolated Vigna radiata and V.mungo seedlings was investigated by pulse nuclear magnetic resonance(NMR) spectroscopy. Various lengths of chilling exposures duringa cool-warm cycle between 20 and 0?C (below 10?C, about 4 h)for the T₁ hysteresis measurement did not cause any visibleinjury symptoms in hypocotyls. However, the profiles of T₁ hysteresisvaried as a result of different chilling exposures. The sumsof the T₁ ratio (for detail see Introduction) reflecting T₁prolongation or shortening upon the warming process were a goodquantitative index for the extent of T₁ hysteresis, and thewide dispersion of this value ranging on the "minus" side (T₁prolongation upon warming) suggested the occurrence of a primaryresponse of cells to chilling stress before obvious visiblesymptoms occur while the T₁ ratio sums on the "plus" side (T₁shortening upon warming) corresponded to a response of seriousvisible injury. Therefore, the sums of the T₁ ratio can be usedas a non-destructive diagnostic tool for monitoring the primaryevent of chilling injury when lacking any visible injury symptoms.The data indicate that the critical temperature for the occurrenceof primary response for chilling stress was around 7.5?C forV. radiata and 12.5?C for V. mungo. (Received February 1, 1988; Accepted June 1, 1988)     

Growth Retardant-Induced Changes in Phototropic Reaction of Vigna radiata Seedlings

The effect of growth retardants on phototropism has been studied in mung bean (Vigna radiata) seedlings. Ancymidol, tetcyclacis, and paclobutrazol inhibited phototropism while AMO 1618 and CCC were ineffective. The fluence-response relationships for phototropism of etiolated seedlings were similar to those previously described for monocots and other dicots. Ancymidol caused a shift in the maximum phototropic response to higher fluence of light. It is suggested that ancymidol may affect phototropism through an effect on the photoreceptor system.     

Nuclear Magnetic Resonance Transverse Relaxation Times of Water Protons in Skeletal Muscle

The observation of the spin-echo decay in a long time domain has revealed that there exist at least three different fractions of non- (or slowly) exchanging water in the rat gastrocnemius muscle. These fractions of water are characterized with different nuclear magnetic resonance (NMR) relaxation times and are identified with the different parts of tissue water. The water associated with the macromolecules was found to be approximately 8% of the total tissue water and not to exchange rapidly with the rest of the intracellular water. The transverse relaxation time (T₂) of the myoplasm is 45 ms which is roughly a 40-fold reduction from that of a dilute electrolyte solution. This fraction of water accounts for 82% of the tissue water. The reduced relaxation time is shown neither to be caused by fast exchange between the hydration and myoplasmic water nor by the diffusion of water across the local magnetic field gradients which arise from the heterogeneity in the sample. About 10% of the tissue water was resolved to be associated with the extracellular space, the relaxation time of which is approximately four times that of the myoplasm. Mathematical treatments of the proposed mechanisms which may be responsible for the reduction of tissue water relaxation times are given in this paper. The results of our study are consistent with the notion that the structure and/or motions of all or part of the cellular water are affected by the macromolecular interface and this causes a change in the NMR relaxation rates.     

Structural Changes in Radish Seedlings Exposed to Cadmium

Radish (Raphanus sativus L. cv. Redondo Vermelho) seedlings were analysed by light and scanning electron microscopy to characterize the structural changes caused by the exposure to 0.5 or 1.0 mM cadmium chloride for 24, 48 and 72 h. The analyses showed changes in the anatomical and morphological characteristics of roots, stems and leaves of two-week-old seedlings. In all tissues, pressure potential was decreased. Premature death with the disintegration of the epidermis and an increase in the number of root hairs was observed in roots exposed to Cd. The stem of seedlings exposed to Cd exhibited more cells layers in the cambial region. The main effects observed in leaves in response to Cd were stomatal closure, lack of cell wall thickening and alterations in the shape of the chloroplasts. It is suggested that the structural changes observed in seedlings treated with Cd were mainly caused by a Cd-induced decrease in water uptake.     

Alleviation of NaCl Stress by Pretreatment with Phytohormones in Vigna radiata

Efficiency of pretreatment as foliar spray of indole-3-acetic acid, gibberellic acid and kinetin, each ranging from 0.1 to 10.0 μM concentration, in restoring the metabolic alterations imposed by NaCl salinity was investigated in Vigna radiata (L.) Wilczek. Glycolate oxidase, superoxide dismutase, catalase and peroxidase activities increased under stress in leaves and roots also. Malondialdehyde content and total peroxide content also increased under stress. All the three hormones used were able to overcome to variable extents the adverse effects of stress imposed by NaCl to these parameters. This revised version was published online in July 2006 with corrections to the Cover Date.     

Estimation of Chilling Sensitivity and Injury in Gloxinia Leaves by the Thermal Hysteresis of NMR Relaxation Times of Water Protons

Thermal hysteresis of longitudinal relaxation times (T₁) ofwater protons in leaf tissues of gloxinia was investigated bypulse nuclear magnetic resonance (NMR) spectroscopy. The profilesof T₁ hysteresis during a slow cool-warm cycle (20 to 0?C) variedwith the degree of chilling injury. General trends of T₁ changesupon warming were as follows: (1) a small increase of T₁ wasobserved when no chilling injury occurred or the degree of injurywas minor, (2) a large increase of T₁ occurred when injury increased,(3) a large decrease of T₁ occurred from the earlier stage ofwarming when serious injury occured. The sum of T₁ ratio (theratio obtained from the difference between the T₁ value in thecooling process and that of the wanning process for eight stepsof cool-warm cycle, divided by the former) reflecting T₁ increaseor decrease during the warming process was related to varietaldifference and seasonal changes of chilling sensitivity. Therefore,T₁ hysteresis can be used as a diagnostic tool in detectingthe chilling sensitivity and the degree of chilling injury. (Received August 27, 1986; Accepted February 24, 1987)     

Properties of Plasma Membrane Isolated from Chilling-Sensitive Etiolated Seedlings of Vigna radiata L

Plasma membrane was isolated in a uniform population and with a high purity from chilling-sensitive etiolated young seedlings of Vigna radiata (mung bean) utilizing an aqueous two polymer phase separation system and subsequent sucrose density gradient. The isolated plasma membrane was associated with vanadate-sensitive and KNO₃-insensitive ATPase. The ATPase has high specificities both for substrate and Mg²⁺ ion with optimum pH at 6.5. It was slightly stimulated by monovalent anions, especially Cl⁻. Proton ionophores such as gramicidin D and carbonyl cyanide p-trifluoromethoxyphenylhydrazone did not stimulate the enzyme activity. The ATPase is apparently latent and highly stimulated by the addition of detergents such as Triton X-100. A maximum stimulation was achieved by the addition of 0.02% Triton X-100. After treatment with proteinase K in an isotonic buffer solution, the enzyme activity was less affected, whereas the peptides were specifically digested. Based on these facts, the isolated plasma membrane vesicles appear to be tightly sealed and in a right-side-out orientation. The plasma membrane ATPase had two inflection points at higher (18.9°C) and lower (6.7°C) temperatures on the Arrhenius plots of the activity. The lower inflection temperature apparently coincided with that of the anisotropy parameter of embedded 1,6-diphenyl-1,3,5-hexatriene, indicating that the membrane bound ATPase activity was affected by a phase transition of membrane lipids and/or temperature-dependent conformational changes in the enzyme molecules per se. Considering the fact that the plant material used here is highly sensitive to chilling temperatures and injured severely by exposure to temperatures below 5°C for a relatively short period, the thermotropic properties of membrane molecules are considered to be involved in the mechanism of chilling injury.     

Isolation and Characterization of Tonoplast from Chilling-Sensitive Etiolated Seedlings of Vigna radiata L

Tonoplasts were isolated in a high purity from etiolated young seedlings of Vigna radiata L. (mung bean) utilizing a sucrose density gradient system. The excised hypocotyls were homogenized in a sorbitol-buffer system and the 3,600 to 156,000g pellets obtained after the differential centrifugations were suspended in a sorbitol medium and loaded on a linear sucrose density gradient. After centrifugation at 89,000g for 2 hours, tonoplasts were banded at the sample load/sucrose interface. Assessed by electron microscopy and marker enzymes, the purity and the quantity were found to be sufficient for biochemical and biophysical analyses. The tonoplasts were associated with NO₃⁻-sensitive and vana-date-insensitive ATPase. The tonoplast ATPase was stimulated by proton ionophores such as carbonyl cyanide p-trifluoromethoxyphenyl-hydrazone and gramicidin D, suggesting a proton-pumping enzyme. In the presence of ATP and Mg²⁺, a proton gradient was formed in the isolated tonoplast vesicles as assessed by fluorescence quenching of quinacrine. The tonoplasts contained several kinds of mannosylated or glycosylated glycoproteins and a major protein (65 kilodaltons) which was unique to the membranes.     

Transverse Relaxation Time of Leaf Water Protons and Membrane Injury in Wheat (Triticum aestivum L.) in Response to High Temperature

The relationship between high temperature stress injury andtemperature dependence of the transverse relaxation time (T₂)of leaf water was examined using NMR in four cultivars of wheatdiffering in their sensitivity to high temperature stress. TheT₂declined with increasing temperature between 25 and 35 °C.A comparison of relative injury based on electrolyte leakageand T₂, between 40 and 50 °C, indicated that while membranepermeability increased with increasing temperature there wasan increase in T₂until 44 and 48 °C in susceptible and tolerantcultivars respectively, followed by a sharp decline. This patternof change in T₂with increasing temperature was consistent whetherthe same or different samples were used for each treatment temperature.Loss of temperature dependence of T₂after heat killing indicatedirreversible changes in T₂, probably due to the loss of membraneintegrity. Heat tolerant varieties, which suffered less membraneinjury, had a higher T₂compared to susceptible varieties. Tolerantvarieties also maintained the T₂of leaf water protons to highertemperatures than did sensitive varieties. This NMR-based, non-invasive,rapid technique could be used to efficiently detect heat injuryin leaf tissues. Copyright 1999 Annals of Botany Company Membrane integrity, transverse relaxation time, high temperature stress, Triticum aestivum L.     

Nano-Boehmite Induced Oxidative and Nitrosative Stress Responses in Vigna radiata L.

Journal of Plant Growth Regulation - Wide spectrum and increasing use of nano-sized aluminum oxyhydroxide (boehmite, nBhm) particles have left a risk of their environmental exposure and...     

Mimosine Mitigates Oxidative Stress in Selenium Deficient Seedlings of Vigna radiata. Part II: Mitochondrial Uptake of 75Selenium and Mimosine

During the growth of selenium (Se)-deficient seedlings of Vigna radiata, exposure to mimosine [2-amino-3-(3-hydroxy-4-oxo-1H-pyridin-1-yl)-propanoic acid], a nonprotein plant amino acid, effectively mitigated stress at 0.1 mM, as reflected in enhancement of growth and efficiency of mitochondrial functions. Since the changes in the seedlings elicited by exposure to mimosine were similar to those effected by Se at an optimal exposure level of 0.75 ppm (Sreekala et al., Biol Trace Elem Res 70:193–207, 1999), the uptake of Se and that of mimosine itself was individually studied in the respiring mitochondria of Se-deficient seedlings (−Se-stressed group) in comparison with those exposed to mimosine during growth at 0.1 mM (Mim 0.1 group). In both groups, the mitochondrial uptake of ⁷⁵Se at 10 μM added increased linearly up to 2 min, attaining steady-state levels thereafter. Uptake levels were 2.3-fold higher in the Mim 0.1 group than in the −Se-stressed group. Double-reciprocal plots of mitochondrial ⁷⁵Se uptake against 2–20 μM in the medium were nonlinear and negative cooperative effects during the uptake were confirmed by Scatchard plots, whereas Hill coefficients were 0.8 and 0.85 for the two groups. Mitochondrial uptake of mimosine, at added levels of 25 or 50 μM, increased linearly up to 1 min and decelerated thereafter. Initial uptake levels of mimosine at 1 min were higher by 6.5-fold at 25 μM and 4-fold at 50 μM in the Mim 0.1 group than those in the −Se-stressed group. Initial uptake levels with added mimosine up to 50 or 100 μM yielded nonlinear double-reciprocal plots; and kinetic analyses at 5 to 50 μM revealed the prevalence of positive cooperativity in the −Se-stressed group and negative cooperativity in the Mim 0.1 group. Involvement of active thiol groups in the uptake of both Se and mimosine were indicated by inhibition studies. Evidence presented for mimosine mediated increase in mitochondrial Se uptake and cooperative interactions thereof underscores the metabolic significance of mimosine.     

Factors Affecting the Prolongation of NMR Relaxation Times of Water Protons in Leaves of Woody Plants Affected by Formation of Insect Galls

Changes in NMR relaxation times (T₁) of water protons and watercontents of leaves of woody plants affected by formation ofinsect galls were studied in Machilus, Zelkova and Cinnamomumparasitized with a gall-midge, an aphid and two different Triozinepsyllids, respectively. The presence of galls in Machilus leavesincreased both T₁ and water contents in the galled leaf tissues,while such tissues in Zelkova showed only increases in T₁. Similartrends for both parameters were also observed in gall-bearingleaf tissues of Machilus and Cinnamomum, with galls caused bytwo different psyllids. It seems that it is the particular characteristicsof leaf tissues of the host plant that determine whether thesystemic effect of the presence of galls is reflected both inT₁ and in water content, or only in T₁. Histologic features,including the presence of tannins in and leakage of electrolytesfrom these materials, were compared with those of normal (ungalled)leaves to determine possible causative factors involved in theprolongation of T₁ relaxation times that were associated withthe presence of insect galls. The eco-physiological implicationof tannins with respect to the host-parasite relationship isalso discussed. (Received October 27, 1989; Accepted April 24, 1990)     

Effect of Phytohormone Pretreatment on Nitrogen Metabolism in Vigna radiata Under Salt Stress

Application of NaCl (electrical conductivity 4.0 mS cm^–1) resulted in about 52, 50 and 55 % reduction in total nitrogen contents in mung bean [Vigna radiata (L.) Wilczek] leaf, root and nodule, respectively. In nodule, nitrogenase activity was reduced by about 84 % under stress as compared with the control set. Glutamine synthetase activity was reduced by about 31, 16 and 23 %, glutamate oxoglutarate aminotransferase activity was reduced by 78, 57 and 42 % and glutamate dehydrogenase activity was reduced by 9, 8 and 42 % in leaf, root and nodule, respectively, under salt stress. The pretreatment with indole-3-acetic acid, gibberellic acid and kinetin, each ranging from 0.1 to 10 µM, in restoring the metabolic alterations imposed by NaCl salinity was investigated in mung bean. The three phytohormones used were able to overcome to variable extents the adverse effects of stress imposed by NaCl solution.     

氧化胁迫下稻苗体内积累的脯氨酸的抗氧化作用

水稻幼苗在外源·OH和1O2氧化胁迫下,体内脯氨酸明显积累,而和H2O2处理对稻苗体内脯氨酸含量无影响。强光亦能诱导稻苗体内脯氨酸积累。脯氨酸预处理则显著抑制·OH、1O2及强光所诱导的稻苗膜脂过氧化作用。在活性氧的产生／检测系统中,脯氨酸对·OH和1O2所引发反应有明显的竞争效应,但对和H2O2的作用没有形响。这些都表明,氧化胁迫下稻苗体内积累的脯氨酸具有抗氧化作用;脯氨酸对活性氧的清除有一定的专一性,即只对·OH和1O2有明显的清除活性。     

The Role of Phytochrome in Anthocyanin Synthesis in Seedlings of Vigna radiata (L.) Wilczek

Previously, it has been demonstrated that the red light-inducedanthocyanin accumulation in mung bean seedlings is mediatedby phytochrome [Dumortier and Vendrig Z. Pflanzenphysiol. 87:313 (1978)]. In this paper the importance of phytochrome forthe accumulation of anthocyanins in seedlings of mung beanswas studied in non-irradiated seedlings and in seedlings irradiatedwith 5 min R. A short FR-irradiation given early after sowing reduced theamount of anthocyanins which were normally found in non-irradiatedseedlings. This indicates that P_FR may be important for at leastpart of the anthocyanin synthesis in the dark. As for the redlight-mediated anthocyanin accumulation, irradiation appearedto be most effective when given to seedlings at the age of 36–48hr. Although the seedlings were sensitive to red light irradiationbefore that time, they were not able to synthesize anthocyaninsuntil they had reached the age of 36 hr. Complete escape ofred/far-red reversibility occurred only when far-red was given12 hr after red, although partial escape could be observed witha shorter time-interval. Furthermore, the time-course of anthocyaninaccumulation after a two-fold R-irradiation was compared withthe effect of a single R-exposure. From the results could beconcluded that the pattern of anthocyanin accumulation is dependenton the time during which P_FR is present in the seedlings. Theseexperiments also indicate that P_FR not only plays a role inthe synthesis of anthocyanins but probably also in their degradation. The results of our study show that phytochrome is importantfor anthocyanin accumulation in non-irradiated mung bean seedlingsas well as in R-irradiated, and that it probably is also involvedin the degradation of the pigment. (Received January 18, 1982; Accepted April 30, 1982)     

Temperature Tolerances of North American Freshwater Fishes Exposed to Dynamic Changes in Temperature

Traditionally lower and upper temperature tolerances of fishes have been quantified in the laboratory via three different experimental approaches: the Fry or incipient lethal temperature (ILT), critical thermal (CTM) and chronic lethal (CLM) methodologies. Although these three experimental laboratory approaches generate endpoints which are quantitatively expressed as a temperature, are determined experimentally with random samples of fish acclimated to specific temperatures, and involve both time and temperature as major test variables, they do not quantify the same response. All three approaches generate valuable, albeit different, information concerning the temperature tolerance of a species. In this review we have summarized published research concerning the tolerance of North American freshwater fishes to dynamic changes in temperature, i.e., tolerance is tested by methods that gradually change temperatures until biological stress is observed. We found more than 450 individual temperature tolerances listed in 80 publications which present original dynamic temperature tolerance data for 116 species, 7 subspecies and 7 hybrids from 19 families of North American freshwater fishes. This total represents about 1/3 of the families and 1/6 of the known North American freshwater species. Temperature tolerance data were partitioned by experimental approach, i.e., critical thermal method (CTM) and chronic lethal method (CLM), and direction of temperature change. Although both CTM and CLM expose fish to dynamic changes in water temperature, these two methods differ in temperature change rates and test endpoints, and hence measure different aspects of thermal stress. A majority of the 80 studies employed CTM to assess temperature tolerance, in particular determination of CTmaxima. One or more CTmaxima has been reported for 108 fishes. Twenty-two fishes have reported highest CTmaxima of 40°C or higher. Several species in the family Cyprinodontidae have generated some of the highest CTmaxima reported for any ectothermic vertebrate. For a variety of reasons, data concerning tolerance of low temperatures are less plentiful. Low temperature tolerance quantified as either CTminima or CLminima were found for a total of 37 fishes. Acclimation temperature exerts a major effect on the temperature tolerance of most North American fish species and it is usually strongly linearly related to both CTmaxima and CTminima. Although we uncovered dynamic temperature tolerance data for 130 fishes, only a single dynamic, temperature tolerance polygon has been published, that for the sheepshead minnow, Cyprinodon variegatus.     

不同浓度NaCl胁迫处理下豇豆幼苗抗氧化酶活性的变化

本研究主要探讨不同浓度NaCl胁迫处理下豇豆(Vigna unguiculata Linn.)幼苗叶片抗氧化酶活性的变化情况.研究结果表明,在0～250 mmol/L NaCl胁迫下,随着盐浓度的增加,豇豆幼苗叶片可溶性蛋白质、脯氨酸和丙二醛含量逐渐增加,在150 mmol/L浓度时,3者的含量都达到最大值;而超氧化物歧化酶(SOD)、过氧化物酶(POD)和过氧化氢酶(CAT)的活性开始逐渐上升,它们的活性分别在100 mmol/L、150 mmol/L和150 mmol/L时达到最大值,然后逐渐下降.同时,对NaCl胁迫下3种抗氧化酶基因的表达进行适时定量PCR分析,分析结果显示3种抗氧化酶基因的转录表达与酶活性的变化一致.说明在不同浓度的NaCl胁迫下,NaCl诱导了sod、pod和cat 3种抗氧化酶基因的表达,3种抗氧化酶活性相应地提高,从而提高了豇豆应对NaCl胁迫的能力.本文结果将为今后豇豆在盐碱栽培生产提供一定的参考.     

Biosynthesis of Phosphatidylglycerol in Isolated Mitochondria of Etiolated Mung Bean (Vigna radiata L.) Seedlings

Phosphatidylglycerophosphate synthase (sn-glycerol-3-phosphate:CDP-diacylglycerol phosphatidyltransferase) and phosphatidylglycerophosphate phosphatase were characterized in mung bean (Vigna radiata L.) mitochondria. The synthase has a rather broad pH optimum between 7 and 9, whereas the phosphatase has one of about 7. Both enzymic activities are stimulated by Triton X-100 and require divalent cations but differ in their cation specificities. The synthase shows apparent Km values of 9 and 3 [mu]M for sn-glycerol-3-phosphate and CDP-diacylglycerol, respectively. Phosphatidylglycerophosphate, in contrast to lysophosphatidic and phosphatidic acid, is effectively dephosphorylated by the phosphatase, which exhibits an apparent Km value of 12 [mu]M for its substrate. Each enzyme shows higher activities with the dipalmitoyl species of its substrate than with the dioleoyl species. These substrate specificities of both enzymes are predominantly based on differences in apparent Vmax values.     

Composition of Bleeding Sap in Vigna radiata

Bleeding sap and nodules from Vigna radiata were analysed for their free amino nitrogen content and amino acid composition at different stages of growth and development. The bleeding sap contained mostly basic amino acids, whereas the nodules contained both acidic and basic amino acids. The amino nitrogen content of the bleeding sap increased during growth and then declined appreciably during fruit development. In contrast, nodule amino nitrogen declined from seedling stage onwards till flowering, increased during fruit development and then declined again. Nitrate reductase activity in the leaves examined at different stages of development increased from seedling stage onwards and was maximum during early fruit-development stage. It declined during pod-filling stage. The study suggests that the amount of nitrogen fixed from the atmosphere is insufficient, so that the plant has to draw upon soil nitrogen as well. This may be necessary due to the high demand of nitrogen during pod filling.